Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

PubMed Central

Matsuda, K; Buckingham, S D; Freeman, J C; Squire, M D; Baylis, H A; Sattelle, D B

1998-01-01

Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit. PMID:9504393

Acetylcholine affects osteocytic MLO-Y4 cells via acetylcholine receptors.

PubMed

Ma, Yuanyuan; Li, Xianxian; Fu, Jing; Li, Yue; Gao, Li; Yang, Ling; Zhang, Ping; Shen, Jiefei; Wang, Hang

2014-03-25

The identification of the neuronal control of bone remodeling has become one of the many significant recent advances in bone biology. Cholinergic activity has recently been shown to favor bone mass accrual by complex cellular regulatory networks. Here, we identified the gene expression of the muscarinic and nicotinic acetylcholine receptors (m- and nAChRs) in mice tibia tissue and in osteocytic MLO-Y4 cells. Acetylcholine, which is a classical neurotransmitter and an osteo-neuromediator, not only influences the mRNA expression of the AChR subunits but also significantly induces the proliferation and viability of osteocytes. Moreover, acetylcholine treatment caused the reciprocal regulation of RANKL and OPG mRNA expression, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes via acetylcholine receptors. The expression of neuropeptide Y and reelin, which are two neurogenic markers, was also modulated by acetylcholine via m- and nAChRs in MLO-Y4 cells. These results indicated that osteocytic acetylcholine receptors might be a new valuable mediator for cell functions and even for bone remodeling. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

alpha 4 beta 2 subunit combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

PubMed

Zwart, R; Abraham, D; Oortgiesen, M; Vijverberg, H P

1994-08-22

Pharmacological characteristics of native neuronal nicotinic acetylcholine receptor-mediated ion currents in mouse N1E-115 neuroblastoma cells have been investigated by superfusion of voltage clamped cells with known concentrations of the agonists acetylcholine, nicotine and cytisine, and the antagonists alpha-bungarotoxin and neuronal bungarotoxin. The sensitivity of the nicotinic acetylcholine receptor for agonists followed the agonist potency rank-order: nicotine approximately acetylcholine > cytisine. The EC50 values of acetylcholine and nicotine are 78 microM and 76 microM, respectively. Equal concentrations of acetylcholine and nicotine induce inward currents with approximately the same peak amplitude whereas cytisine induces much smaller inward currents. Acetylcholine-induced currents are unaffected by high concentrations of alpha-bungarotoxin. Conversely, at 10 and 90 nM neuronal bungarotoxin reduces the amplitude of the 1 mM acetylcholine-induced inward current to 47% and 11% of control values, respectively. Both the agonist potency rank-order and the differential sensitivity to snake toxins of nicotinic receptors in N1E-115 cells are consistent with the known pharmacological profile of alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes and distinct from those of all other nicotinic acetylcholine receptors of known functional subunit compositions. All data indicate that the native nicotinic acetylcholine receptor in N1E-115 cells is an assembly of alpha 4 and beta 2 subunits, the putative major subtype of nicotinic acetylcholine receptor in the brain.

Effect of α₇ nicotinic acetylcholine receptor agonists and antagonists on motor function in mice.

PubMed

Welch, Kevin D; Pfister, James A; Lima, Flavia G; Green, Benedict T; Gardner, Dale R

2013-02-01

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChRs located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The toxicity and teratogenicity of many plants (which results in millions of dollars in losses annually to the livestock industry) are due to various toxins that bind to nAChRs including deltaline and methyllycaconitine (MLA) from larkspur (Delphinium) species, and nicotine and anabasine from tobacco (Nicotiana) species. The primary result of the actions of these alkaloids at nAChRs is neuromuscular paralysis and respiratory failure. The objective of this study was to further characterize the motor coordination deficiencies that occur upon exposure to a non-lethal dose of nAChR antagonists MLA and deltaline as well as nAChR agonists nicotine and anabasine. We evaluated the effect of nAChR agonists and antagonists on the motor function and coordination in mice using a balance beam, grip strength meter, rotarod, open field analysis and tremor monitor. These analyses demonstrated that within seconds after treatment the mice had significant loss of motor function and coordination that lasted up to 1 min, followed by a short period of quiescence. Recovery to normal muscle coordination was rapid, typically within approximately 10 min post-dosing. However, mice treated with the nAChR agonist nicotine and anabasine required a slightly longer time to recover some aspects of normal muscle function in comparison to mice treated with the nAChR antagonist MLA or deltaline. Published by Elsevier Inc.

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

PubMed Central

Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

2015-01-01

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill.

PubMed

González, Alfredo; Crittenden, Elizabeth L; García, Dana M

2004-07-13

In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

Heterogeneity of binding of muscarinic receptor antagonists in rat brain homogenates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, J.H.; el-Fakahany, E.E.

1985-06-01

The binding properties of (-)-(/sup 3/H)quinuclidinyl benzilate and (/sup 3/H) N-methylscopolamine to muscarinic acetylcholine receptors have been investigated in rat brain homogenates. The binding of both antagonists demonstrated high affinity and saturability. Analysis of the binding data resulted in linear Scatchard plots. However, (-)-(/sup 3/H)quinuclidinyl benzilate showed a significantly higher maximal binding capacity than that of (/sup 3/H)N-methylscopolamine. Displacement of both ligands with several muscarinic receptor antagonists resulted in competition curves in accordance with the law of mass-action for quinuclidinyl benzilate, atropine and scopolamine. A similar profile was found for the quaternary ammonium analogs of atropine and scopolamine when (/supmore » 3/H)N-methylscopolamine was used to label the receptors. However, when these hydrophilic antagonists were used to displace (-)-(/sup 3/H) quinuclidinyl benzilate binding, they showed interaction with high- and low-affinity binding sites. On the other hand, the nonclassical muscarinic receptor antagonist, pirenzepine, was able to displace both ligands from two binding sites. The present data are discussed in terms of the relationship of this anomalous heterogenity of binding of these hydrophilic muscarinic receptor antagonists and the proposed M1 and M2 receptor subtypes.« less

The dual orexin receptor antagonist, DORA-22, lowers histamine levels in the lateral hypothalamus and prefrontal cortex without lowering hippocampal acetylcholine.

PubMed

Yao, Lihang; Ramirez, Andres D; Roecker, Anthony J; Fox, Steven V; Uslaner, Jason M; Smith, Sean M; Hodgson, Robert; Coleman, Paul J; Renger, John J; Winrow, Christopher J; Gotter, Anthony L

2017-07-01

Chronic insomnia is defined as a persistent difficulty with sleep initiation maintenance or non-restorative sleep. The therapeutic standard of care for this condition is treatment with gamma-aminobutyric acid (GABA) A receptor modulators, which promote sleep but are associated with a panoply of side effects, including cognitive and memory impairment. Dual orexin receptor antagonists (DORAs) have recently emerged as an alternative therapeutic approach that acts via a distinct and more selective wake-attenuating mechanism with the potential to be associated with milder side effects. Given their distinct mechanism of action, the current work tested the hypothesis that DORAs and GABA A receptor modulators differentially regulate neurochemical pathways associated with differences in sleep architecture and cognitive performance induced by these pharmacological mechanisms. Our findings showed that DORA-22 suppresses the release of the wake neurotransmitter histamine in the lateral hypothalamus, prefrontal cortex, and hippocampus with no significant alterations in acetylcholine levels. In contrast, eszopiclone, commonly used as a GABA A modulator, inhibited acetylcholine secretion across brain regions with variable effects on histamine release depending on the extent of wakefulness induction. In normal waking rats, eszopiclone only transiently suppressed histamine secretion, whereas this suppression was more obvious under caffeine-induced wakefulness. Compared with the GABA A modulator eszopiclone, DORA-22 elicits a neurotransmitter profile consistent with wake reduction that does not impinge on neurotransmitter levels associated with cognition and rapid eye movement sleep. © 2017 International Society for Neurochemistry.

Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors.

PubMed

Molgó, Jordi; Marchot, Pascale; Aráoz, Rómulo; Benoit, Evelyne; Iorga, Bogdan I; Zakarian, Armen; Taylor, Palmer; Bourne, Yves; Servent, Denis

2017-08-01

We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

DOE Office of Scientific and Technical Information (OSTI.GOV)

Middleton, R.E.; Cohen, J.B.

1991-07-16

The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with highmore » affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.« less

Effect of a nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

USDA-ARS?s Scientific Manuscript database

Nicotinic acetylcholine receptors (nAChR) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChR located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The...

Muscarinic and nicotinic acetylcholine receptor agonists: current scenario in Alzheimer's disease therapy.

PubMed

Verma, Stuti; Kumar, Ashwini; Tripathi, Timir; Kumar, Awanish

2018-04-16

Alzheimer's disease (AD) has become the primary cause of dementia. It shows a progressive cognitive dysfunction with degenerating neurons. Acetylcholine receptors (AChRs) propagate the cognitive ability and it consists of two primary members namely muscarinic (mAChRs) and nicotinic receptors (nAChRs). Where mAChRs is G-protein coupled receptor, (nAChRs) are ligand-gated ion channels. The conventional therapeutic regimen for AD consists of three acetylcholinestearse inhibitors while a single NMDA receptor antagonist. Researchers around the globe are developing new and modifying the existing AChRs agonists to develop lead candidates with lower risk to benefit ratio where benefits clearly outweigh the adverse events. We have searched PubMed, MEDLINE, Google scholar, Science Direct and, Web of Science with keywords "Muscarinic/Nicotinic acetylcholine receptor, agonists and, AD". The literature search included articles written in English. Scientific relevance for clinical studies, basic science studies is eligibility criteria for articles referred in this paper. M1 is the primary muscarinic subtype while α7 is the primary nAChR subtype that is responsible for cognition and memory and these two have been the major recent experimental targets for mAChR agonist strategy. The last cholinergic receptor agonist to enter phase 3 trial was EVP-6124 (Enceniclin) but was withdrawn due to severe gastrointestinal adverse effects. We aim to present an overview of the efforts and achievements in targeting Muscarinic and Nicotinic acetylcholine receptor in the current review for development of better AD therapeutics. © 2018 Royal Pharmaceutical Society.

Solution structure of {alpha}-conotoxin PIA, a novel antagonist of {alpha}6 subunit containing nicotinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chi, Seung-Wook; Lee, Si-Hyung; Kim, Do-Hyoung

2005-12-30

{alpha}-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing {alpha}6 and {alpha}3 subunits. {alpha}-conotoxin PIA displays 75-fold higher affinity for rat {alpha}6/{alpha}3{beta}2{beta}3 nAChRs than for rat {alpha}3{beta}2 nAChRs. We have determined the three-dimensional structure of {alpha}-conotoxin PIA by nuclear magnetic resonance spectroscopy. The {alpha}-conotoxin PIA has an '{omega}-shaped' overall topology as other {alpha}4/7 subfamily conotoxins. Yet, unlike other neuronally targeted {alpha}4/7-conotoxins, its N-terminal tail Arg{sup 1}-Asp{sup 2}-Pro{sup 3} protrudes out of its main molecular body because Asp{sup 2}-Pro{sup 3}-Cys{sup 4}-Cys{sup 5} forms a stable type I {beta}-turn. In addition, amore » kink introduced by Pro{sup 15} in the second loop of this toxin provides a distinct steric and electrostatic environment from those in {alpha}-conotoxins MII and GIC. By comparing the structure of {alpha}-conotoxin PIA with other functionally related {alpha}-conotoxins we suggest structural features in {alpha}-conotoxin PIA that may be associated with its unique receptor recognition profile.« less

Effect of α{sub 7} nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Welch, Kevin D., E-mail: kevin.welch@ars.usda.gov; Pfister, James A.; Lima, Flavia G.

2013-02-01

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChRs located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The toxicity and teratogenicity of many plants (which results in millions of dollars in losses annually to the livestock industry) are due to various toxins that bind to nAChRs including deltaline and methyllycaconitine (MLA) from larkspur (Delphinium) species, and nicotine and anabasine from tobacco (Nicotiana) species. The primary result of the actions of these alkaloids at nAChRs is neuromuscularmore » paralysis and respiratory failure. The objective of this study was to further characterize the motor coordination deficiencies that occur upon exposure to a non-lethal dose of nAChR antagonists MLA and deltaline as well as nAChR agonists nicotine and anabasine. We evaluated the effect of nAChR agonists and antagonists on the motor function and coordination in mice using a balance beam, grip strength meter, rotarod, open field analysis and tremor monitor. These analyses demonstrated that within seconds after treatment the mice had significant loss of motor function and coordination that lasted up to 1 min, followed by a short period of quiescence. Recovery to normal muscle coordination was rapid, typically within approximately 10 min post-dosing. However, mice treated with the nAChR agonist nicotine and anabasine required a slightly longer time to recover some aspects of normal muscle function in comparison to mice treated with the nAChR antagonist MLA or deltaline. -- Highlights: ► Mice treated with nAChR agonists and antagonists have a loss in motor function. ► These deficits are temporary as near normal motor function returns within 10 min. ► There are compound-specific differences in the effects on motor function.« less

Acetylcholine receptor antibody

MedlinePlus

... found in the blood of most people with myasthenia gravis . The antibody affects a chemical that sends signals ... Performed This test is used to help diagnose myasthenia gravis . Normal Results Normally, there is no acetylcholine receptor ...

Brain nicotinic acetylcholine receptors are involved in stress-induced potentiation of nicotine reward in rats.

PubMed

Javadi, Parastoo; Rezayof, Ameneh; Sardari, Maryam; Ghasemzadeh, Zahra

2017-07-01

The aim of the present study was to examine the possible role of nicotinic acetylcholine receptors of the dorsal hippocampus (CA1 regions), the medial prefrontal cortex or the basolateral amygdala in the effect of acute or sub-chronic stress on nicotine-induced conditioned place preference. Our results indicated that subcutaneous administration of nicotine (0.2 mg/kg) induced significant conditioned place preference. Exposure to acute or sub-chronic elevated platform stress potentiated the response of an ineffective dose of nicotine. Pre-conditioning intra-CA1 (0.5-4 µg/rat) or intra-medial prefrontal cortex (0.2-0.3 µg/rat) microinjection of mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist) reversed acute stress-induced potentiation of nicotine reward as measured in the conditioned place preference paradigm. By contrast, pre-conditioning intra-basolateral amygdala microinjection of mecamylamine (4 µg/rat) potentiated the effects of acute stress on nicotine reward. Our findings also showed that intra-CA1 or intra-medial prefrontal cortex, but not intra-basolateral amygdala, microinjection of mecamylamine (4 µg/rat) prevented the effect of sub-chronic stress on nicotine reward. These findings suggest that exposure to elevated platform stress potentiates the rewarding effect of nicotine which may be associated with the involvement of nicotinic acetylcholine receptors. It seems that there is a different contribution of the basolateral amygdala, the medial prefrontal cortex or the CA1 nicotinic acetylcholine receptors in stress-induced potentiation of nicotine-induced conditioned place preference.

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

PubMed Central

Gill, JasKiran K.; Savolainen, Mari; Young, Gareth T.; Zwart, Ruud; Sher, Emanuele; Millar, Neil S.

2011-01-01

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular “orthosteric” binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ∼200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site. PMID:21436053

Melatonin modulates rat myotube-acetylcholine receptors by inhibiting calmodulin.

PubMed

de Almeida-Paula, Lidiana Duarte; Costa-Lotufo, Leticia V; Silva Ferreira, Zulma; Monteiro, Amanda Elisa G; Isoldi, Mauro Cesar; Godinho, Rosely O; Markus, Regina P

2005-11-21

Melatonin, the pineal gland hormone, modulates alpha-bungarotoxin sensitive nicotinic acetylcholine receptors in sympathetic nerve terminals, cerebellum and chick retina imposing a diurnal variation in functional responses [Markus, R.P., Zago, W.M., Carneiro, R.C., 1996. Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 279, 18-22; Markus, R.P., Santos, J.M., Zago, W., Reno, L.A., 2003. Melatonin nocturnal surge modulates nicotinic receptors and nicotine-induced [3HI] glutamate release in rat cerebellum slices. J. Pharmacol. Exp. Ther. 305, 525-530; Sampaio, L.F.S., Hamassaki-Britto, D.E., Markus, R.P., 2005. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells. Braz. J. Med. Biol. Res. 38, 603-613]. Here we show that in rat myotubes forskolin and melatonin reduced the number of nicotinic acetylcholine receptors expressed in plasma membrane. In addition, these cells expressed melatonin MT1 receptors, which are known to be coupled to G(i)-protein. However, the pharmacological profile of melatonin analogs regarding the reduction in cyclic AMP accumulation and number of nicotinic acetylcholine receptors did not point to a mechanism mediated by activation of G(i)-protein coupled receptors. On the other hand, calmidazolium, a classical inhibitor of calmodulin, reduced in a similar manner both effects. Considering that one isoform of adenylyl cyclase present in rat myotubes is regulated by Ca2+/calmodulin, we propose that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation.

Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries.

PubMed

Tangsucharit, Panot; Takatori, Shingo; Zamami, Yoshito; Goda, Mitsuhiro; Pakdeechote, Poungrat; Kawasaki, Hiromu; Takayama, Fusako

2016-01-01

The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

PubMed

Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M; DeSimone, John A; Lyall, Vijay

2015-01-01

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine

PubMed Central

Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M.; DeSimone, John A.; Lyall, Vijay

2015-01-01

Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol. PMID:26039516

Beta-phenylethylamine stimulates striatal acetylcholine release through activation of the AMPA glutamatergic pathway.

PubMed

Ishida, Kota; Murata, Mikio; Kato, Masatoshi; Utsunomiya, Iku; Hoshi, Keiko; Taguchi, Kyoji

2005-09-01

Using an in vivo intra-striatal microdialysis technique, we examined the effects of systemically administered beta-phenylethylamine (beta-PEA), a psychomotor stimulating trace amine, on striatal acetylcholine release in freely moving rats. Infusion of N-methyl-D-aspartic acid (NMDA; 10(-5) M) significantly increased acetylcholine release. In addition, locally applied amino-3-hydroxy-5-methylisozasole-4-propionic acid (AMPA; 10(-5) M) significantly increased acetylcholine release in the striatum. Intra-striatal application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M), an AMPA-type glutamatergic receptor antagonist, had little effect on acetylcholine release, while application of MK-801 (10(-5) M, 10(-6) M), an NMDA-type glutamatergic receptor antagonist, significantly reduced acetylcholine release. Acetylcholine within striatal perfusate was significantly increased by intraperitoneal administration of beta-PEA in a dose-dependent manner. This increase in acetylcholine release was completely blocked by application of CNQX (10(-5) M) through the microdialysis probe into the striatum. However, increased acetylcholine response to systemic beta-PEA was unaltered by addition of MK-801 to the perfusion medium. These results suggest a regulatory function of beta-PEA, mediated by AMPA-type glutamatergic receptors, on the release of acetylcholine in the rat striatum.

Progesterone Modulates a Neuronal Nicotinic Acetylcholine Receptor

NASA Astrophysics Data System (ADS)

Valera, S.; Ballivet, M.; Bertrand, D.

1992-10-01

The major brain nicotinic acetylcholine receptor is assembled from two subunits termed α 4 and nα 1. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroid is not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.

Molecular properties of muscarinic acetylcholine receptors

PubMed Central

HAGA, Tatsuya

2013-01-01

Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories. PMID:23759942

IL-1 receptor antagonist-mediated therapeutic effect in murine myasthenia gravis is associated with suppressed serum proinflammatory cytokines, C3, and anti-acetylcholine receptor IgG1.

PubMed

Yang, Huan; Tüzün, Erdem; Alagappan, Dhivyaa; Yu, Xiang; Scott, Benjamin G; Ischenko, Alexander; Christadoss, Premkumar

2005-08-01

In myasthenia gravis (MG), TNF and IL-1beta polymorphisms and high serum levels of these proinflammatory cytokines have been observed. Likewise, TNF and IL-1beta are critical for the activation of acetylcholine receptor (AChR)-specific T and B cells and for the development of experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. We tested the therapeutic effect of human recombinant IL-1 receptor antagonist (IL-1ra) in C57BL/6 mice with EAMG. Multiple daily injections of 0.01 mg of IL-1ra administered for 2 wk following two AChR immunizations decreased the incidence and severity of clinical EAMG. Furthermore, IL-1ra treatment of mice with ongoing clinical EAMG reduced the clinical symptoms of disease. The IL-1ra-mediated suppression of clinical disease was associated with suppressed serum IFN-gamma, TNF-alpha, IL-1beta, IL-2, IL-6, C3, and anti-AChR IgG1 without influencing total serum IgG. Therefore, IL-1ra could be used as a nonsteroidal drug for the treatment of MG.

Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: identification and cellular localization.

PubMed

Elhusseiny, A; Cohen, Z; Olivier, A; Stanimirović, D B; Hamel, E

1999-07-01

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become

Nicotinic Acetylcholine Receptors in Sensory Cortex

ERIC Educational Resources Information Center

Metherate, Raju

2004-01-01

Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

Modeling study of mecamylamine block of muscle type acetylcholine receptors.

PubMed

Ostroumov, Konstantin; Shaikhutdinova, Asya; Skorinkin, Andrey

2008-04-01

The blocking action of mecamylamine on different types of nicotinic acetylcholine receptors (nAChRs) has been extensively studied and used as a tool to characterize the nAChRs from different synapses. However, mechanism of mecamylamine action was not fully explored for all types of nAChRs. In the present study, we provide brief description of the mecamylamine action on muscle nAChRs expressed at the frog neuromuscular junction. In this preparation mecamylamine block of nAChRs was accompanied by a use-dependent block relief induced by membrane depolarization combined with the activation of nAChRs by endogenous agonist acetylcholine (ACh). Further, three kinetic models of possible mecamylamine interaction with nAChRs were analyzed including simple open channel block, symmetrical trapping block and asymmetrical trapping block. This analysis suggested that mecamylamine action could be described on the basis of trapping mechanism, when the antagonist remained inside the channel even in the absence of bound agonist. Such receptors with trapped mecamylamine inside were predicted to have a closing rate constant about three times faster than resting one and a fast voltage-dependent unblocking rate constant. Specific experimental conditions and morphological organization of the neuromuscular synapses were considered to simulate time course of the mecamylamine block development. Thus, likewise for the neuronal nAChRs, the trapping mechanism determined the action of mecamylamine on synaptic neuromuscular currents evoked by the endogenous agonist acetylcholine (ACh), however specific morphological organization of the synaptic transmission delayed time development of the currents block.

Acetylcholine protects mesenteric arteries against hypoxia/reoxygenation injury via inhibiting calcium-sensing receptor.

PubMed

Zhao, Ming; He, Xi; Yang, Yong-Hua; Yu, Xiao-Jiang; Bi, Xue-Yuan; Yang, Yang; Xu, Man; Lu, Xing-Zhu; Sun, Qiang; Zang, Wei-Jin

2015-04-01

The Ca(2+)-sensing receptor (CaSR) plays an important role in regulating vascular tone. In the present study, we investigated the positive effects of the vagal neurotransmitter acetylcholine by suppressing CaSR activation in mesenteric arteries exposed to hypoxia/reoxygenation (H/R). The artery rings were exposed to a modified 'ischemia mimetic' solution and an anaerobic environment to simulate an H/R model. Our results showed that acetylcholine (10(-6) mol/L) significantly reduced the contractions induced by KCl and phenylephrine and enhanced the endothelium-dependent relaxation induced by acetylcholine. Additionally, acetylcholine reduced CaSR mRNA expression and activity when the rings were subjected to 4 h of hypoxia and 12 h of reoxygenation. Notably, the CaSR antagonist NPS2143 significantly reduced the contractions but did not improve the endothelium-dependent relaxation. When a contractile response was achieved with extracellular Ca(2+), both acetylcholine and NPS2143 reversed the H/R-induced abnormal vascular vasoconstriction, and acetylcholine reversed the calcimimetic R568-induced abnormal vascular vasoconstriction in the artery rings. In conclusion, this study suggests that acetylcholine ameliorates the dysfunctional vasoconstriction of the arteries after H/R, most likely by decreasing CaSR expression and activity, thereby inhibiting the increase in intracellular calcium concentration. Our findings may be indicative of a novel mechanism underlying ACh-induced vascular protection. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

EVP-6124, a novel and selective α7 nicotinic acetylcholine receptor partial agonist, improves memory performance by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors.

PubMed

Prickaerts, Jos; van Goethem, Nick P; Chesworth, Richard; Shapiro, Gideon; Boess, Frank G; Methfessel, Christoph; Reneerkens, Olga A H; Flood, Dorothy G; Hilt, Dana; Gawryl, Maria; Bertrand, Sonia; Bertrand, Daniel; König, Gerhard

2012-02-01

EVP-6124, (R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide, is a novel partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs) that was evaluated here in vitro and in vivo. In binding and functional experiments, EVP-6124 showed selectivity for α7 nAChRs and did not activate or inhibit heteromeric α4β2 nAChRs. EVP-6124 had good brain penetration and an adequate exposure time. EVP-6124 (0.3 mg/kg, p.o.) significantly restored memory function in scopolamine-treated rats (0.1 mg/kg, i.p.) in an object recognition task (ORT). Although donepezil at 0.1 mg/kg, p.o. or EVP-6124 at 0.03 mg/kg, p.o. did not improve memory in this task, co-administration of these sub-efficacious doses fully restored memory. In a natural forgetting test, an ORT with a 24 h retention time, EVP-6124 improved memory at 0.3 mg/kg, p.o. This improvement was blocked by the selective α7 nAChR antagonist methyllycaconitine (0.3 mg/kg, i.p. or 10 μg, i.c.v.). In co-application experiments of EVP-6124 with acetylcholine, sustained exposure to EVP-6124 in functional investigations in oocytes caused desensitization at concentrations greater than 3 nM, while lower concentrations (0.3-1 nM) caused an increase in the acetylcholine-evoked response. These actions were interpreted as representing a co-agonist activity of EVP-6124 with acetylcholine on α7 nAChRs. The concentrations of EVP-6124 that resulted in physiological potentiation were consistent with the free drug concentrations in brain that improved memory performance in the ORT. These data suggest that the selective partial agonist EVP-6124 improves memory performance by potentiating the acetylcholine response of α7 nAChRs and support new therapeutic strategies for the treatment of cognitive impairment. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'. Copyright © 2011 Elsevier Ltd. All rights reserved.

Nicotine evokes kinetic tremor by activating the inferior olive via α7 nicotinic acetylcholine receptors.

PubMed

Kunisawa, Naofumi; Iha, Higor A; Shimizu, Saki; Tokudome, Kentaro; Mukai, Takahiro; Kinboshi, Masato; Serikawa, Tadao; Ohno, Yukihiro

2016-11-01

Nicotinic acetylcholine (nACh) receptors are implicated in the pathogenesis of movement disorders (e.g., tremor) and epilepsy. Here, we performed behavioral and immunohistochemical studies using mice and rats to elucidate the mechanisms underlying nicotine-induced tremor. Treatments of animals with nicotine (0.5-2mg/kg, i.p.) elicited kinetic tremor, which was completely suppressed by the nACh receptor antagonist mecamylamine (MEC). The specific α7 nACh receptor antagonist methyllycaconitine (MLA) also inhibited nicotine-induced tremor, whereas the α4β2 nACh antagonist dihydro-β-erythroidine (DHβE) or the peripheral α3β4 nACh antagonist hexamethonium showed no effects. Mapping analysis of Fos protein expression, a biological marker of neural excitation, revealed that a tremorgenic dose (1mg/kg) of nicotine region-specifically elevated Fos expression in the piriform cortex (PirC), medial habenula, solitary nucleus and inferior olive (IO) among 44 brain regions examined. In addition, similarly to the tremor responses, nicotine-induced Fos expression in the PirC and IO was selectively antagonized by MLA, but not by DHβE. Furthermore, an electrical lesioning of the IO, but not the PirC, significantly suppressed the induction of nicotine tremor. The present results suggest that nicotine elicits kinetic tremor in rodents by activating the IO neurons via α7 nACh receptors. Copyright © 2016 Elsevier B.V. All rights reserved.

Relationships between chemical structure and affinity for acetylcholine receptors

PubMed Central

Abramson, F. B.; Barlow, R. B.; Mustafa, M. G.; Stephenson, R. P.

1969-01-01

1. Series of analogues of acetylcholine have been prepared in which the acetyl group was replaced by phenylacetyl, cyclohexylacetyl, diphenylacetyl, dicyclohexylacetyl, (±)-phenylcyclohexylacetyl, benziloyl and (±)-phenylcyclohexylhydroxyacetyl groups and the trimethylammonium group was replaced by Me2EtN+, MeEt2N+, Et3N+, [Formula: see text] Further series were prepared in which the acetoxyethyl group was replaced by ethoxyethyl, phenylethoxyethyl, cyclohexylethoxyethyl, diphenylethoxyethyl, and dicyclohexylethoxyethyl groups, and by n-pentyl, 5-phenylpentyl, 5-cyclohexylpentyl and 5:5-diphenylpentyl groups. 2. The ethoxyethyl and n-pentyl series contain some compounds which are agonists or partial agonists when tested on the isolated guinea-pig ileum, but all the other compounds are antagonists. 3. The affinity of the compounds for the postganglionic (“muscarinesensitive”) acetylcholine receptors has been measured in conditions in which the antagonists have been shown to be acting competitively. There were considerable differences between their affinities, the most active (log K, 9·8) having one million times the affinity of the least active (log K, 3·7). 4. The changes in affinity as the onium group was modified were not entirely independent of changes in the rest of the molecule. Increasing the size of the onium group, as judged from conductivity measurements on simpler onium salts, increased affinity in the series containing one large group (phenyl or cyclohexyl) but, in the series with two large groups, affinity declined when the size was increased beyond -+NMeEt2. 5. In general, the effects of changes in the rest of the molecule on affinity were bigger than the effects of changes in the onium group and there were bigger interactions. Affinity was increased to a greater extent by introducing one phenyl and one cyclohexyl group together than by introducing either two phenyl or two cyclohexyl groups; the increment was greater than the separate

Adenosine A1 Receptors in Mouse Pontine Reticular Formation Depress Breathing, Increase Anesthesia Recovery Time, and Decrease Acetylcholine Release

PubMed Central

Gettys, George C.; Liu, Fang; Kimlin, Ed; Baghdoyan, Helen A.; Lydic, Ralph

2012-01-01

Background Clinical and preclinical data demonstrate the analgesic actions of adenosine. Central administration of adenosine agonists, however, suppresses arousal and breathing by poorly understood mechanisms. This study tested the two-tailed hypothesis that adenosine A1 receptors in the pontine reticular formation (PRF) of C57BL/6J mice modulate breathing, behavioral arousal, and PRF acetylcholine release. Methods Three sets of experiments used 51 mice. First, breathing was measured by plethysmography after PRF microinjection of the adenosine A1 receptor agonist N6-sulfophenyl adenosine (SPA) or saline. Second, mice were anesthetized with isoflurane and time to recovery of righting response (RoRR) was quantified after PRF microinjection of SPA or saline. Third, acetylcholine release in the PRF was measured before and during microdialysis delivery of SPA, the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), or SPA and DPCPX. Results First, SPA significantly decreased respiratory rate (−18%), tidal volume (−12%) and minute ventilation (−16%). Second, SPA concentration accounted for 76% of the variance in RoRR. Third, SPA concentration accounted for a significant amount of the variance in acetylcholine release (52%), RoRR (98%), and breathing rate (86%). DPCPX alone caused a concentration-dependent increase in acetylcholine, decrease in RoRR, and decrease in breathing rate. Coadministration of SPA and DPCPX blocked the SPA-induced decrease in acetylcholine and increase in RoRR. Conclusions Endogenous adenosine acting at adenosine A1 receptors in the PRF modulates breathing, behavioral arousal, and acetylcholine release. The results support the interpretation that an adenosinergic-cholinergic interaction within the PRF comprises one neurochemical mechanism underlying the wakefulness stimulus for breathing. PMID:23263018

Antibronchospastic activity of MEN10,627, a novel tachykinin NK2 receptor antagonist, in guinea-pig airways.

PubMed

Perretti, F; Ballati, L; Manzini, S; Maggi, C A; Evangelista, S

1995-01-24

The antibronchospastic activity against acetylcholine, antigen, histamine plus platelet-activating factor (PAF) or the selective tachykinin neurokinin (NK)1 and NK2 receptor agonists of the novel tachykinin NK2 receptor antagonist, MEN10,627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta)), was studied in anesthetized guinea-pigs. MEN10,627 (30-100 nmol/kg i.v.) reduced in a dose-dependent manner the bronchospasm induced by the tachykinin NK2 receptor agonist [beta Ala8]neurokinin A-(4-10) and the effect of the highest dose lasted up to 5 h from its administration. Conversely, airway constriction induced by the NK1 receptor agonist [Sar9]substance P sulfone or acetylcholine was unaffected by MEN10,627 up to a dose of 3 mumol/kg i.v. In animals sensitized with ovalbumin and pretreated with the endopeptidase inhibitor phosphoramidon, the aerosolized antigen produced a bronchospasm which was inhibited by MEN10,627 (30-100 nmol/kg i.v.) but not by the tachykinin NK1 receptor antagonist, (+/-)-CP96,345 ([2R,3R-cis- and [2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine]) (3 mumol/kg i.v.). Both MEN10,627 (30-100 nmol/kg i.v.) and (+/-)-CP96,345 (30-300 nmol/kg i.v.) reduced the PAF-induced hyperresponsiveness to histamine, without affecting the hypotension induced by PAF or the bronchospasm induced by histamine in guinea-pigs not exposed to PAF, showing the involvement of both tachykinin NK1 and NK2 receptors in this model. In summary, MEN10,627 behaves as a potent, selective and long-lasting tachykinin NK2 receptor antagonist in vivo. Further, tachykinin NK2 receptors could be activated during allergic responses and in the development of airway hyperresponsiveness.

A computational model of the nicotinic acetylcholine binding site

NASA Astrophysics Data System (ADS)

Gálvez-ruano, Enrique; Iriepa-Canalda, Isabel; Morreale, Antonio; Lipkowitz, Kenny B.

1999-01-01

We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro-β-erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).

In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat.

PubMed

Killi, Uday K; Wsol, Vladimir; Soukup, Ondrej; Kuca, Kamil; Winder, Michael; Tobin, Gunnar

2014-02-01

Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime. © 2013 Wiley Publishing Asia Pty Ltd.

Adenosine A(1) receptors in mouse pontine reticular formation depress breathing, increase anesthesia recovery time, and decrease acetylcholine release.

PubMed

Gettys, George C; Liu, Fang; Kimlin, Ed; Baghdoyan, Helen A; Lydic, Ralph

2013-02-01

Clinical and preclinical data demonstrate the analgesic actions of adenosine. Central administration of adenosine agonists, however, suppresses arousal and breathing by poorly understood mechanisms. This study tested the two-tailed hypothesis that adenosine A1 receptors in the pontine reticular formation (PRF) of C57BL/6J mice modulate breathing, behavioral arousal, and PRF acetylcholine release. Three sets of experiments used 51 mice. First, breathing was measured by plethysmography after PRF microinjection of the adenosine A1 receptor agonist N-sulfophenyl adenosine (SPA) or saline. Second, mice were anesthetized with isoflurane and the time to recovery of righting response (RoRR) was quantified after a PRF microinjection of SPA or saline. Third, acetylcholine release in the PRF was measured before and during microdialysis delivery of SPA, the adenosine A1 receptor antagonist 1, 3-dipropyl-8-cyclopentylxanthine, or SPA and 1, 3-dipropyl-8-cyclopentylxanthine. First, SPA significantly decreased respiratory rate (-18%), tidal volume (-12%), and minute ventilation (-16%). Second, SPA concentration accounted for 76% of the variance in RoRR. Third, SPA concentration accounted for a significant amount of the variance in acetylcholine release (52%), RoRR (98%), and breathing rate (86%). 1, 3-dipropyl-8-cyclopentylxanthine alone caused a concentration-dependent increase in acetylcholine, a decrease in RoRR, and a decrease in breathing rate. Coadministration of SPA and 1, 3-dipropyl-8-cyclopentylxanthine blocked the SPA-induced decrease in acetylcholine and increase in RoRR. Endogenous adenosine acting at adenosine A1 receptors in the PRF modulates breathing, behavioral arousal, and acetylcholine release. The results support the interpretation that an adenosinergic-cholinergic interaction within the PRF comprises one neurochemical mechanism underlying the wakefulness stimulus for breathing.

Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

PubMed

Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

2016-01-15

After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. Copyright © 2015 Elsevier B.V. All rights reserved.

Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia and airway mucus formation in vitro and in vivo

PubMed Central

Gundavarapu, Sravanthi; Wilder, Julie A.; Mishra, Neerad C.; Rir-sima-ah, Jules; Langley, Raymond J.; Singh, Shashi P.; Saeed, Ali Imran; Jaramillo, Richard J.; Gott, Katherine M.; Peña-Philippides, Juan Carlos; Harrod, Kevin S.; McIntosh, J. Michael; Buch, Shilpa; Sopori, Mohan L.

2012-01-01

Background Airway mucus hypersecretion is a key pathophysiological feature in number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. Objectives Characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. Methods IL-13 and gamma-aminobutyric acid receptors (GABAARs) are implicated in airway mucus. We examined the role of IL-13 and GABAARs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells, and secondhand cigarette smoke and/or ovalbumin-induced mucus formation in vivo. Results Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABAAR antagonist picrotoxin (PIC). Airway epithelial cells express α7/α9/α10 nicotinic acetylcholine receptors (nAChRs) and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells and/or in mouse airways. Conclusions Nicotine-induced airway mucus formation is independent of IL-13 and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various pro-mucoid agents, including IL-13. In the absence of nicotine, acetylcholine may be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABAARα2 in the MUC5AC pathway. Acetylcholine and α-7-nAChRs may serve as therapeutic targets to control airway mucus. PMID:22578901

The pharmacological rationale for combining muscarinic receptor antagonists and β-adrenoceptor agonists in the treatment of airway and bladder disease☆

PubMed Central

Dale, Philippa R; Cernecka, Hana; Schmidt, Martina; Dowling, Mark R; Charlton, Steven J; Pieper, Michael P; Michel, Martin C

2014-01-01

Muscarinic receptor antagonists and β-adrenoceptor agonists are used in the treatment of obstructive airway disease and overactive bladder syndrome. Here we review the pharmacological rationale for their combination. Muscarinic receptors and β-adrenoceptors are physiological antagonists for smooth muscle tone in airways and bladder. Muscarinic agonism may attenuate β-adrenoceptor-mediated relaxation more than other contractile stimuli. Chronic treatment with one drug class may regulate expression of the target receptor but also that of the opposing receptor. Prejunctional β2-adrenoceptors can enhance neuronal acetylcholine release. Moreover, at least in the airways, muscarinic receptors and β-adrenoceptors are expressed in different locations, indicating that only a combined modulation of both systems may cause dilatation along the entire bronchial tree. While all of these factors contribute to a rationale for a combination of muscarinic receptor antagonists and β-adrenoceptor agonists, the full value of such combination as compared to monotherapy can only be determined in clinical studies. PMID:24682092

AMPA Receptors Mediate Acetylcholine Release from Starburst Amacrine Cells in the Rabbit Retina

PubMed Central

FIRTH, SALLY I.; LI, WEI; MASSEY, STEPHEN C.; MARSHAK, DAVID W.

2012-01-01

The light response of starburst amacrine cells is initiated by glutamate released from bipolar cells. To identify the receptors that mediate this response, we used a combination of anatomical and physiological techniques. An in vivo, rabbit eyecup was preloaded with [3H]-choline, and the [3H]-acetylcholine (ACh) released into the superfusate was monitored. A photopic, 3 Hz flashing light increased ACh release, and the selective AMPA receptor antagonist, GYKI 53655, blocked this light-evoked response. Nonselective AMPA/kainate agonists increased the release of ACh, but the specific kainate receptor agonist, SYM 2081, did not increase ACh release. Selective AMPA receptor antagonists, GYKI 53655 or GYKI 52466, also blocked the responses to agonists. We conclude that the predominant excitatory input to starburst amacrine cells is mediated by AMPA receptors. We also labeled lightly fixed rabbit retinas with antisera to choline acetyltransferase (ChAT), AMPA receptor subunits GluR1, GluR2/3, or GluR4, and kainate receptor subunits GluR6/7 and KA2. Labeled puncta were observed in the inner plexiform layer with each of these antisera to glutamate receptors, but only GluR2/3-IR puncta and GluR4-IR puncta were found on the ChAT-IR processes. The same was true of starburst cells injected intracellularly with Neurobiotin, and these AMPA receptor subunits were localized to two populations of puncta. The AMPA receptors are expected to desensitize rapidly, enhancing the sensitivity of starburst amacrine cells to moving or other rapidly changing stimuli. PMID:14515241

Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists.

PubMed

Cheng, Han; Lear-Rooney, Calli M; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W; Olinger, Gene G; Rong, Lijun

2015-10-01

Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious

Inhibition of Ebola and Marburg Virus Entry by G Protein-Coupled Receptor Antagonists

PubMed Central

Cheng, Han; Lear-Rooney, Calli M.; Johansen, Lisa; Varhegyi, Elizabeth; Chen, Zheng W.; Olinger, Gene G.

2015-01-01

ABSTRACT Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of

Activation of α7 nicotinic acetylcholine receptors persistently enhances hippocampal synaptic transmission and prevents Aß-mediated inhibition of LTP in the rat hippocampus.

PubMed

Ondrejcak, Tomas; Wang, Qinwen; Kew, James N C; Virley, David J; Upton, Neil; Anwyl, Roger; Rowan, Michael J

2012-02-29

Nicotinic acetylcholine receptors mediate fast cholinergic modulation of glutamatergic transmission and synaptic plasticity. Here we investigated the effects of subtype selective activation of the α7 nicotinic acetylcholine receptors on hippocampal transmission and the inhibition of synaptic long-term potentiation by the Alzheimer's disease associated amyloid ß-protein (Aß). The α7 nicotinic acetylcholine receptor agonist "compound A" ((R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl))thiophene-2-carboxamide) induced a rapid-onset persistent enhancement of synaptic transmission in the dentate gyrus in vitro. Consistent with a requirement for activation of α7 nicotinic acetylcholine receptors, the type II α7-selective positive allosteric modulator PheTQS ((3aR, 4S, 9bS)-4-(4-methylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) potentiated, and the antagonist methyllycaconitine (MLA) prevented the persistent enhancement. Systemic injection of the agonist also induced a similar MLA-sensitive persistent enhancement of synaptic transmission in the CA1 area in vivo. Remarkably, although compound A did not affect control long-term potentiation (LTP) in vitro, it prevented the inhibition of LTP by Aß1-42 and this effect was inhibited by MLA. These findings strongly indicate that activation of α7 nicotinic acetylcholine receptors is sufficient to persistently enhance hippocampal synaptic transmission and to overcome the inhibition of LTP by Aß. Copyright © 2011 Elsevier B.V. All rights reserved.

NMDA receptor antagonists inhibit catalepsy induced by either dopamine D1 or D2 receptor antagonists.

PubMed

Moore, N A; Blackman, A; Awere, S; Leander, J D

1993-06-11

In the present study, we investigated the ability of NMDA receptor antagonists to inhibit catalepsy induced by haloperidol, or SCH23390 and clebopride, selective dopamine D1 and D2 receptor antagonists respectively. Catalepsy was measured by recording the time the animal remained with its forepaws placed over a rod 6 cm above the bench. Pretreatment with either the non-competitive NMDA receptor antagonist, MK-801 (0.25-0.5 mg/kg i.p.) or the competitive antagonist, LY274614 (10-20 mg/kg i.p.) reduced the cataleptic response produced by haloperidol (10 mg/kg), SCH23390 (2.5-10 mg/kp i.p.) or clebopride (5-20 mg/kg i.p.). This demonstrates that NMDA receptor antagonists will reduce both dopamine D1 and D2 receptor antagonist-induced catalepsy. Muscle relaxant doses of chlordiazepoxide (10 mg/kg i.p.) failed to reduce the catalepsy induced by haloperidol, suggesting that the anticataleptic effect of the NMDA receptor antagonists was not due to a non-specific action. These results support the hypothesis that NMDA receptor antagonists may have beneficial effects in disorders involving reduced dopaminergic function, such as Parkinson's disease.

Chemokine receptor antagonists: part 2.

PubMed

Pease, James E; Horuk, Richard

2009-02-01

The first part of this two-part review discussed approaches to generating antagonists for some of the CC chemokine receptors, including CCR1, CCR2, CCR3, and CCR4. This second part of the series concludes the review by describing antagonists for CCR5, CCR8, CCR9, CXCR3, CXCR4, and promiscuous antagonists. Chemokine receptor antagonists have found mixed success as therapeutics. Although one antagonist--maraviroc, a CCR5 inhibitor to treat AIDS--has been registered as an approved drug, this is the only success so far. There have been many failures in the clinic and we discuss the idea of promiscuous receptor antagonists as an alternative approach.

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

PubMed

Joseph, Lauren; Thomsen, Morgane

2017-06-30

Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

Back to the future: Rational maps for exploring acetylcholine receptor space and time.

PubMed

Tessier, Christian J G; Emlaw, Johnathon R; Cao, Zhuo Qian; Pérez-Areales, F Javier; Salameh, Jean-Paul J; Prinston, Jethro E; McNulty, Melissa S; daCosta, Corrie J B

2017-11-01

Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties. Copyright © 2017 Elsevier B.V. All rights reserved.

The role of 5 HT6-receptor antagonists in Alzheimer's disease: an update.

PubMed

Khoury, Rita; Grysman, Noam; Gold, Jake; Patel, Kush; Grossberg, George T

2018-06-01

Despite recent advances in Alzheimer's disease (AD) research, no breakthrough treatments have been discovered. Cholinesterase inhibitors and the NMDA-receptor antagonist memantine are currently the two approved symptomatic treatments for AD. 5-HT6 receptor antagonism has recently emerged as a promising treatment strategy to improve cognition in AD, with a modest side-effect profile. Areas covered: 5-HT6 receptors, exclusively found in the central nervous system, modulate primarily GABA and glutamate levels, facilitating the secondary release of other neurotransmitters including dopamine, noradrenaline, and acetylcholine, all of which are compromised in AD. This review discusses findings of preclinical and phase I-III clinical trials conducted with three major 5-HT6 receptor antagonists: idalopirdine, intepirdine, and SUVN-502, in the field of AD. Expert opinion: Despite early positive findings, larger phase-III trials have failed to demonstrate any statistically significant impact on cognition for both idalopirdine and intepirdine, as adjunct to cholinesterase inhibitors. Paradoxically, 5-HT6 receptor agonists have also been shown to have cognitive enhancing properties. Thus, a better understanding of the mechanism of action of the 5-HT6 receptor and its ligands is warranted. Investigating 5-HT6 receptor partial or inverse agonists may be promising in future AD trials.

Mortality Benefit of Recombinant Human Interleukin-1 Receptor Antagonist for Sepsis Varies by Initial Interleukin-1 Receptor Antagonist Plasma Concentration.

PubMed

Meyer, Nuala J; Reilly, John P; Anderson, Brian J; Palakshappa, Jessica A; Jones, Tiffanie K; Dunn, Thomas G; Shashaty, Michael G S; Feng, Rui; Christie, Jason D; Opal, Steven M

2018-01-01

Plasma interleukin-1 beta may influence sepsis mortality, yet recombinant human interleukin-1 receptor antagonist did not reduce mortality in randomized trials. We tested for heterogeneity in the treatment effect of recombinant human interleukin-1 receptor antagonist by baseline plasma interleukin-1 beta or interleukin-1 receptor antagonist concentration. Retrospective subgroup analysis of randomized controlled trial. Multicenter North American and European clinical trial. Five hundred twenty-nine subjects with sepsis and hypotension or hypoperfusion, representing 59% of the original trial population. Random assignment of placebo or recombinant human interleukin-1 receptor antagonist × 72 hours. We measured prerandomization plasma interleukin-1 beta and interleukin-1 receptor antagonist and tested for statistical interaction between recombinant human interleukin-1 receptor antagonist treatment and baseline plasma interleukin-1 receptor antagonist or interleukin-1 beta concentration on 28-day mortality. There was significant heterogeneity in the effect of recombinant human interleukin-1 receptor antagonist treatment by plasma interleukin-1 receptor antagonist concentration whether plasma interleukin-1 receptor antagonist was divided into deciles (interaction p = 0.046) or dichotomized (interaction p = 0.028). Interaction remained present across different predicted mortality levels. Among subjects with baseline plasma interleukin-1 receptor antagonist above 2,071 pg/mL (n = 283), recombinant human interleukin-1 receptor antagonist therapy reduced adjusted mortality from 45.4% to 34.3% (adjusted risk difference, -0.12; 95% CI, -0.23 to -0.01), p = 0.044. Mortality in subjects with plasma interleukin-1 receptor antagonist below 2,071 pg/mL was not reduced by recombinant human interleukin-1 receptor antagonist (adjusted risk difference, +0.07; 95% CI, -0.04 to +0.17), p = 0.230. Interaction between plasma interleukin-1 beta concentration and recombinant human

Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

PubMed

Bourdin, Céline M; Lebreton, Jacques; Mathé-Allainmat, Monique; Thany, Steeve H

2015-08-15

From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 μM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 μM and 39.3 μM whereas LMA10203 and zacopride possessed IC50 values of 8.07 μM and 7.04 μM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Differential effects of subtype-specific nicotinic acetylcholine receptor agonists on early and late hippocampal LTP.

PubMed

Kroker, Katja S; Rast, Georg; Rosenbrock, Holger

2011-12-05

Brain nicotinic acetylcholine receptors are involved in several neuropsychiatric disorders, e.g. Alzheimer's and Parkinson's diseases, Tourette's syndrome, schizophrenia, depression, autism, attention deficit hyperactivity disorder, and anxiety. Currently, approaches selectively targeting the activation of specific nicotinic acetylcholine receptors are in clinical development for treatment of memory impairment of Alzheimer's disease patients. These are α4β2 and α7 nicotinic acetylcholine receptor agonists which are believed to enhance cholinergic and glutamatergic neurotransmission, respectively. In order to gain a better insight into the mechanistic role of these two nicotinic acetylcholine receptors in learning and memory, we investigated the effects of the α4β2 nicotinic acetylcholine receptor agonist TC-1827 and the α7 nicotinic acetylcholine receptor partial agonist SSR180711 on hippocampal long-term potentiation (LTP), a widely accepted cellular experimental model of memory formation. Generally, LTP is distinguished in an early and a late form, the former being protein-synthesis independent and the latter being protein-synthesis dependent. TC-1827 was found to increase early LTP in a bell-shaped dose dependent manner, but did not affect late LTP. In contrast, the α7 nicotinic acetylcholine receptor partial agonist SSR180711 showed enhancing effects on both early and late LTP in a bell-shaped manner. Furthermore, SSR180711 not only increased early LTP, but also transformed it into late LTP, which was not observed with the α4β2 nicotinic acetylcholine receptor agonist. Therefore, based on these findings α7 nicotinic acetylcholine receptor (partial) agonists appear to exhibit stronger efficacy on memory improvement than α4β2 nicotinic acetylcholine receptor agonists. Copyright © 2011 Elsevier B.V. All rights reserved.

Neuronal nicotinic receptor antagonist reduces anxiety-like behavior in mice.

PubMed

Roni, Monzurul Amin; Rahman, Shafiqur

2011-10-31

Brain cholinergic neurotransmission has been implicated in the modulation of anxiety in humans and evidence suggests that drugs targeting neuronal nicotinic acetylcholine receptor (nAChR) could have potential for the treatment of anxiety. The objective of present study was to examine anxiolytic effects of lobeline (0.04 or 0.1 mg/kg), a nAChR antagonist, in C57BL/6J mice using elevated plus-maze (EPM) and marble-burying test. Lobeline (0.04 mg/kg) significantly increased open arm time on EPM and reduced number of marbles buried. Similarly, mecamylamine (0.3 mg/kg) produced anxiolytic effects, while peripherally acting hexamethonium (0.3 mg/kg) failed to produce any response. These results provide evidence that lobeline has anxiolytic potential and nAChR antagonists may represent a new class of anxiolytics in humans. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Supavilai, P.; Karobath, M.

1985-02-04

GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with /sup 3/H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 ..mu..M. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BRmore » agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table.« less

Neuropeptide S attenuates neuropathological, neurochemical and behavioral changes induced by the NMDA receptor antagonist MK-801

PubMed Central

Okamura, Naoe; Reinscheid, Rainer K.; Ohgake, Shintaro; Iyo, Masaomi; Hashimoto, Kenji

2009-01-01

Neuropeptide S (NPS) and its cognate receptor were reported to mediate anxiolytic-like and arousal effects. NPS receptors are predominantly expressed in the brain, especially in limbic structures, including amygdala, olfactory nucleus, subiculum and retrosplenial cortex. In contrast, the NPS precursor is expressed in only a few brainstem nuclei where it is co-expressed with various excitatory transmitters, including glutamate. The current study investigates interactions of the NPS system with glutamatergic neurotransmission. It has been suggested that dysfunctions in glutamatergic neurotransmission via N-methyl-D-aspartate (NMDA) receptors might be involved in the pathophysiology of schizophrenia since NMDA receptor antagonists, such as MK-801, have been shown to induce psychotic-like behavior in humans and animal models. Also, MK-801 is known to produce histological changes such as cytoplasmic vacuoles in retrosplenial cortex neurons where NPS receptors are highly expressed. In this study we show that NPS is able to alleviate neuropathological, neurochemical and behavioral changes produced by NMDA receptor antagonists. NPS treatment attenuated MK-801-induced vacuolization in the rat retrosplenial cortex in a dose dependent manner that can be blocked by an NPS receptor-selective antagonist. NPS also suppressed MK-801-induced increases of extracellular acetylcholine levels in the retrosplenial cortex. In the prepulse inhibition (PPI) assay, animals pretreated with NPS recovered significantly from MK-801-induced disruption of PPI. Our study suggests that NPS may have protective effects against the neurotoxic and behavioral changes produced by NMDA receptor antagonists and that NPS receptor agonists may elicit antipsychotic effects. PMID:19576911

Pharmacology of JB-9315, a new selective histamine H2-receptor antagonist.

PubMed

Palacios, B; Montero, M J; Sevilla, M A; San Román, L

1998-02-01

1. The histamine H2-receptor antagonistic activity and antisecretory and antiulcer effects of JB-9315 were studied in comparison with the standard H2 blocker ranitidine. 2. In vitro, JB-9315 is a competitive antagonist of histamine H2 receptors in the isolated, spontaneously beating guinea-pig right atrium, with a pA2 value of 7.30 relative to a value of 7.36 for ranitidine. JB-9315 was specific for the histamine H2 receptor because, at high concentration, it did not affect histamine- or acetylcholine-induced contractions in guinea-pig isolated ileum or rat isolated duodenum, respectively. 3. JB-9315 dose dependently inhibited histamine-, pentagastrin- or carbachol-stimulated acid secretion and basal secretion in the perfused stomach preparation of the anesthetized rat. In the pylorus-ligated rat after intraperitoneal administration, total acid output over 4 h was inhibited by JB-9315 with an ID50 of 32.8 mg/kg, confirming its H2-receptor antagonist properties. 4. JB-9315 showed antiulcer activity against cold stress plus indomethacin-induced lesions with an ID50 of 6.8 mg/kg. 5. JB-9315, 50 and 100 mg/kg, inhibited macroscopic gastric hemorrhagic lesions induced by ethanol. In contrast, ranitidine (50 mg/kg) failed to reduce these lesions. 6. These results indicate that JB-9315 is a new antiulcer drug that exerts a cytoprotective effect in addition to its gastric antisecretory activity.

Rivastigmine improves isolation rearing-induced prepulse inhibition deficits via muscarinic acetylcholine receptors in mice.

PubMed

Higashino, Kosuke; Ago, Yukio; Umeki, Takahiro; Hasebe, Shigeru; Onaka, Yusuke; Hashimoto, Hitoshi; Takuma, Kazuhiro; Matsuda, Toshio

2016-02-01

The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice. We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice. Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured. Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors. Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.

Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

NASA Astrophysics Data System (ADS)

Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

1987-11-01

A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

PubMed

Navarria, Andrea; Wohleb, Eric S; Voleti, Bhavya; Ota, Kristie T; Dutheil, Sophie; Lepack, Ashley E; Dwyer, Jason M; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S

2015-10-01

Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. Copyright © 2015 Elsevier Inc. All rights reserved.

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice.

PubMed

Bazalakova, M H; Wright, J; Schneble, E J; McDonald, M P; Heilman, C J; Levey, A I; Blakely, R D

2007-07-01

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.

The challenges of modulating the 'rest and digest' system: acetylcholine receptors as drug targets.

PubMed

VanPatten, Sonya; Al-Abed, Yousef

2017-01-01

Acetylcholine, a major neurotransmitter of the parasympathetic and sympathetic nervous systems, was discovered in the early 1900s. Over the years, researchers have revealed much about its regulation, properties of its receptors and features of the downstream signaling that influence its terminal effects. The acetylcholine system, traditionally associated with neuromuscular communication, is now known to play a crucial part in modulation of the immune system and other 'rest and digest' effects. Recent research seeks to elucidate the system's role in brain functions including cognition, sleep, arousal, motivation, reward and pain. We highlight clinically approved and experimental drugs that modulate the acetylcholine receptors. The complexities in targeting the acetylcholine receptors are vast and finding future indications for drug development associated with specific acetylcholine receptors remains a challenge. Copyright © 2016 Elsevier Ltd. All rights reserved.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Transient Receptor Potential Channel Opening Releases Endogenous Acetylcholine, which Contributes to Endothelium-Dependent Relaxation Induced by Mild Hypothermia in Spontaneously Hypertensive Rat but Not Wistar-Kyoto Rat Arteries.

PubMed

Zou, Q; Leung, S W S; Vanhoutte, P M

2015-08-01

Mild hypothermia causes endothelium-dependent relaxations, which are reduced by the muscarinic receptor antagonist atropine. The present study investigated whether endothelial endogenous acetylcholine contributes to these relaxations. Aortic rings of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were contracted with prostaglandin F2 α and exposed to progressive mild hypothermia (from 37 to 31°C). Hypothermia induced endothelium-dependent, Nω-nitro-l-arginine methyl ester-sensitive relaxations, which were reduced by atropine, but not by mecamylamine, in SHR but not in WKY rat aortae. The responses in SHR aortae were also reduced by acetylcholinesterase (the enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake), and vesamicol (inhibitor of acetylcholine release). The mild hypothermia-induced relaxations in both SHR and WKY rat aortae were inhibited by AMTB [N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide; the transient receptor potential (TRP) M8 inhibitor]; only those in SHR aortae were inhibited by HC-067047 [2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide; TRPV4 antagonist] while those in WKY rat aortae were reduced by HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; TRPA1 antagonist]. The endothelial uptake of extracellular choline and release of cyclic guanosine monophosphate was enhanced by mild hypothermia and inhibited by HC-067047 in SHR but not in WKY rat aortae. Compared with WKY rats, the SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but a lesser amount of vesicular acetylcholine transporter, located mainly in the endothelium. Thus, mild hypothermia causes nitric oxide-dependent relaxations by opening TRPA1 channels in WKY rat aortae

Role of the nicotinic acetylcholine receptor in Alzheimer's disease pathology and treatment.

PubMed

Lombardo, Sylvia; Maskos, Uwe

2015-09-01

Alzheimer's Disease (AD) is the major form of senile dementia, characterized by neuronal loss, extracellular deposits, and neurofibrillary tangles. It is accompanied by a loss of cholinergic tone, and acetylcholine (ACh) levels in the brain, which were hypothesized to be responsible for the cognitive decline observed in AD. Current medication is restricted to enhancing cholinergic signalling for symptomatic treatment of AD patients. The nicotinic acetylcholine receptor family (nAChR) and the muscarinic acetylcholine receptor family (mAChR) are the target of ACh in the brain. Both families of receptors are affected in AD. It was demonstrated that amyloid beta (Aβ) interacts with nAChRs. Here we discuss how Aβ activates or inhibits nAChRs, and how this interaction contributes to AD pathology. We will discuss the potential role of nAChRs as therapeutic targets. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. Copyright © 2014 Elsevier Ltd. All rights reserved.

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES✩

PubMed Central

Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

2013-01-01

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5–10 mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2− interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. PMID:22732654

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

PubMed

Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

2014-06-05

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. Copyright © 2012 Elsevier B.V. All rights reserved.

Ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppressing nicotinic acetylcholine receptor-ion channels in cultured bovine adrenal medullary cells.

PubMed

Li, Xiaojia; Toyohira, Yumiko; Horisita, Takafumi; Satoh, Noriaki; Takahashi, Keita; Zhang, Han; Iinuma, Munekazu; Yoshinaga, Yukari; Ueno, Susumu; Tsutsui, Masato; Sata, Takeyoshi; Yanagihara, Nobuyuki

2015-12-01

Ikarisoside A is a natural flavonol glycoside derived from plants of the genus Epimedium, which have been used in Traditional Chinese Medicine as tonics, antirheumatics, and aphrodisiacs. Here, we report the effects of ikarisoside A and three other flavonol glycosides on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that ikarisoside A (1-100 μM), but not icariin, epimedin C, or epimedoside A, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. Ikarisoside A had little effect on catecholamine secretion induced by veratridine and 56 mM K(+). Ikarisoside A (1-100 μM) also inhibited (22)Na(+) influx and (45)Ca(2+) influx induced by acetylcholine in a concentration-dependent manner similar to that of catecholamine secretion. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, ikarisoside A (0.1-100 μM) directly inhibited the current evoked by acetylcholine. It also suppressed (14)C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine at 1-100 μM and 10-100 μM, respectively. The present findings suggest that ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

END-PLATE ACETYLCHOLINE RECEPTOR: STRUCTURE, MECHANISM, PHARMACOLOGY, AND DISEASE

PubMed Central

Sine, Steven M.

2012-01-01

The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction. PMID:22811427

FRET-based sensors for the human M1-, M3-, and M5-acetylcholine receptors.

PubMed

Ziegler, Nicole; Bätz, Julia; Zabel, Ulrike; Lohse, Martin J; Hoffmann, Carsten

2011-02-01

Based on the recently developed approach to generate fluorescence resonance energy transfer (FRET)-based sensors to measure GPCR activation, we generated sensor constructs for the human M(1)-, M(3)-, and M(5)-acetylcholine receptor. The receptors were labeled with cyan fluorescent protein (CFP) at their C-terminus, and with fluorescein arsenical hairpin binder (FlAsH) via tetra-cysteine tags inserted in the third intracellular loop. We then measured FRET between the donor CFP and the acceptor FlAsH in living cells and real time. Agonists like acetylcholine, carbachol, or muscarine activate each receptor construct with half-maximal activation times between 60 and 70ms. Removal of the agonist caused the reversal of the signal. Compared with all other agonists, oxotremorine M differed in two major aspects: it caused significantly slower signals at M(1)- and M(5)-acetylcholine receptors and the amplitude of these signals was larger at the M(1)-acetylcholine receptor. Concentration-response curves for the agonists reveal that all agonists tested, with the mentioned exception of oxotremorine M, caused similar maximal FRET-changes as acetylcholine for the M(1)-, M(3)- and M(5)-acetylcholine receptor constructs. Taken together our data support the notion that orthosteric agonists behave similar at different muscarinic receptor subtypes but that kinetic differences can be observed for receptor activation. Copyright © 2010 Elsevier Ltd. All rights reserved.

Tramadol state-dependent memory: involvement of dorsal hippocampal muscarinic acetylcholine receptors.

PubMed

Jafari-Sabet, Majid; Jafari-Sabet, Ali-Reza; Dizaji-Ghadim, Ali

2016-08-01

The effects on tramadol state-dependent memory of bilateral intradorsal hippocampal (intra-CA1) injections of physostigmine, an acetylcholinesterase inhibitor, and atropine, a muscarinic acetylcholine receptor antagonist, were examined in adult male NMRI mice. A single-trial step-down passive avoidance task was used for the assessment of memory retention. Post-training intra-CA1 administration of an atypical μ-opioid receptor agonist, tramadol (0.5 and 1 μg/mouse), dose dependently impaired memory retention. Pretest injection of tramadol (0.5 and 1 μg/mouse, intra-CA1) induced state-dependent retrieval of the memory acquired under the influence of post-training tramadol (1 μg/mouse, intra-CA1). A pretest intra-CA1 injection of physostigmine (1 μg/mouse) reversed the memory impairment induced by post-training administration of tramadol (1 μg/mouse, intra-CA1). Moreover, pretest administration of physostigmine (0.5 and 1 μg/mouse, intra-CA1) with an ineffective dose of tramadol (0.25 μg/mouse, intra-CA1) also significantly restored retrieval. Pretest administration of physostigmine (0.25, 0.5, and 1 μg/mouse, intra-CA1) by itself did not affect memory retention. A pretest intra-CA1 injection of the atropine (1 and 2 μg/mouse) 5 min before the administration of tramadol (1 μg/mouse, intra-CA1) dose dependently inhibited tramadol state-dependent memory. Pretest administration of atropine (0.5, 1, and 2 μg/mouse, intra-CA1) by itself did not affect memory retention. It can be concluded that dorsal hippocampal muscarinic acetylcholine receptor mechanisms play an important role in the modulation of tramadol state-dependent memory.

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

PubMed

Chiara, David C; Trinidad, Jonathan C; Wang, Dong; Ziebell, Michael R; Sullivan, Deirdre; Cohen, Jonathan B

2003-01-21

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor-induced Sjögren's syndrome-like sialadenitis.

PubMed

Tahara, M; Tsuboi, H; Segawa, S; Asashima, H; Iizuka-Koga, M; Hirota, T; Takahashi, H; Kondo, Y; Matsui, M; Matsumoto, I; Sumida, T

2017-02-01

We showed recently that M3 muscarinic acetylcholine receptor (M3R)-reactive CD3 + T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid-related orphan receptor-gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R -/- ) mice immunized with murine M3R peptide mixture were inoculated into recombination-activating gene 1 knockout (Rag-1 -/- ) mice (M3R -/- →Rag-1 -/- ) with MIS. Immunized M3R -/- mice (pretransfer treatment) and M3R -/- →Rag-1 -/- mice (post-transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide-stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme-linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4 + T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)-γ and interleukin (IL)-17 production significantly compared with phosphate-buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4 + T cells rather than CD11c + cells. Post-transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN-γ and IL-17 production (P < 0·05). In vitro, A213 suppressed IFN-γ and IL-17 production from M3R-stimulated splenocytes and CD4 + T cells of immunized M3R -/- mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL-17 production from Th17 differentiated CD4 + T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS-like sialadenitis through suppression of IL-17 and IFN-γ production

Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

PubMed

Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi

2017-03-01

GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca 2+ influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

Concomitant Release of Ventral Tegmental Acetylcholine and Accumbal Dopamine by Ghrelin in Rats

PubMed Central

Jerlhag, Elisabet; Janson, Anna Carin; Waters, Susanna; Engel, Jörgen A.

2012-01-01

Ghrelin, an orexigenic peptide, regulates energy balance specifically via hypothalamic circuits. Growing evidence suggest that ghrelin increases the incentive value of motivated behaviours via activation of the cholinergic-dopaminergic reward link. It encompasses the cholinergic afferent projection from the laterodorsal tegmental area (LDTg) to the dopaminergic cells of the ventral tegmental area (VTA) and the mesolimbic dopamine system projecting from the VTA to nucleus accumbens (N.Acc.). Ghrelin receptors (GHS-R1A) are expressed in these reward nodes and ghrelin administration into the LDTg increases accumbal dopamine, an effect involving nicotinic acetylcholine receptors in the VTA. The present series of experiments were undertaken directly to test this hypothesis. Here we show that ghrelin, administered peripherally or locally into the LDTg concomitantly increases ventral tegmental acetylcholine as well as accumbal dopamine release. A GHS-R1A antagonist blocks this synchronous neurotransmitter release induced by peripheral ghrelin. In addition, local perfusion of the unselective nicotinic antagonist mecamylamine into the VTA blocks the ability of ghrelin (administered into the LDTg) to increase N.Acc.-dopamine, but not VTA-acetylcholine. Collectively our data indicate that ghrelin activates the LDTg causing a release of acetylcholine in the VTA, which in turn activates local nicotinic acetylcholine receptors causing a release of accumbal dopamine. Given that a dysfunction in the cholinergic-dopaminergic reward system is involved in addictive behaviours, including compulsive overeating and alcohol use disorder, and that hyperghrelinemia is associated with such addictive behaviours, ghrelin-responsive circuits may serve as a novel pharmacological target for treatment of alcohol use disorder as well as binge eating. PMID:23166710

Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

PubMed

Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

2015-06-01

We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors.

Counteracting desensitization of human α7-nicotinic acetylcholine receptors with bispyridinium compounds as an approach against organophosphorus poisoning.

PubMed

Scheffel, Corinna; Niessen, Karin V; Rappenglück, Sebastian; Wanner, Klaus T; Thiermann, Horst; Worek, Franz; Seeger, Thomas

2018-09-01

Irreversible inhibition of acetylcholinesterase (AChE) resulting in accumulation of acetylcholine and overstimulation of muscarinic and nicotinic receptors accounts for the acute toxicity of organophosphorus compounds (OP). Accordingly, the mainstay pharmacotherapy against poisoning by OP comprises the competitive muscarinic acetylcholine receptor antagonist atropine to treat muscarinic effects and, in addition, oximes to reactivate inhibited AChE. A therapeutic gap still remains in the treatment of desensitized nicotinic acetylcholine receptors following OP exposure. Hereby, nicotinic effects result in paralysis of the central and peripheral respiratory system if untreated. Thus, these receptors pose an essential target for therapeutic indication to address these life-threatening nicotinic symptoms of the cholinergic crisis. Identification of ligands regulating dynamic transitions between functional states by binding to modulatory sites appears to be a promising strategy for therapeutic intervention. In this patch clamp study, the ability of differently substituted bispyridinium non-oximes to "resensitize" i.e. to recover the activity of desensitized human homomeric α7-type nAChRs stably transfected in CHO cells was investigated and compared to the already described α7-specific positive allosteric modulator PNU-120596. The structures of these bispyridinium analogues were based on the lead structure of the tert-butyl-substituted bispyridinium propane MB327, which has been shown to have a positive therapeutic effect due to a non-competitive antagonistic action at muscle-type nAChRs in vivo and has been found to have a positive allosteric activity at neuronal receptors in vitro. Prior to test compounds, desensitization of hα7-nAChRs was verified by applying an excess of nicotine revealing activation at low, and desensitization at high concentrations. Thereby, desensitization could be reduced by modulation with PNU-120596. Desensitization was further verified by

Acetylcholine receptors in the human retina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hutchins, J.B.; Hollyfield, J.G.

1985-11-01

Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer ofmore » the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.« less

Synthesis of Selective Agonists for the α7 Nicotinic Acetylcholine Receptor with In Situ Click-Chemistry on Acetylcholine-Binding Protein Templates

PubMed Central

Yamauchi, John G.; Gomez, Kimberly; Grimster, Neil; Dufouil, Mikael; Nemecz, Ákos; Fotsing, Joseph R.; Ho, Kwok-Yiu; Talley, Todd T.; Sharpless, K. Barry; Fokin, Valery V.

2012-01-01

The acetylcholine-binding proteins (AChBPs), which serve as structural surrogates for the extracellular domain of nicotinic acetylcholine receptors (nAChRs), were used as reaction templates for in situ click-chemistry reactions to generate a congeneric series of triazoles from azide and alkyne building blocks. The catalysis of in situ azide-alkyne cycloaddition reactions at a dynamic subunit interface facilitated the synthesis of potentially selective compounds for nAChRs. We investigated compound sets generated in situ with soluble AChBP templates through pharmacological characterization with α7 and α4β2 nAChRs and 5-hydroxytryptamine type 3A receptors. Analysis of activity differences between the triazole 1,5-syn- and 1,4-anti-isomers showed a preference for the 1,4-anti-triazole regioisomers among nAChRs. To improve nAChR subtype selectivity, the highest-potency building block for α7 nAChRs, i.e., 3α-azido-N-methylammonium tropane, was used for additional in situ reactions with a mutated Aplysia californica AChBP that was made to resemble the ligand-binding domain of the α7 nAChR. Fourteen of 50 possible triazole products were identified, and their corresponding tertiary analogs were synthesized. Pharmacological assays revealed that the mutated binding protein template provided enhanced selectivity of ligands through in situ reactions. Discrete trends in pharmacological profiles were evident, with most compounds emerging as α7 nAChR agonists and α4β2 nAChR antagonists. Triazoles bearing quaternary tropanes and aromatic groups were most potent for α7 nAChRs. Pharmacological characterization of the in situ reaction products established that click-chemistry synthesis with surrogate receptor templates offered novel extensions of fragment-based drug design that were applicable to multisubunit ion channels. PMID:22784805

Bisquaternary dimers of strychnine and brucine. A new class of potent enhancers of antagonist binding to muscarinic M2 receptors.

PubMed

Zlotos, D P; Buller, S; Holzgrabe, U; Mohr, K

2003-06-12

Bisquaternary dimers of strychnine and brucine were synthesized and their allosteric effect on muscarinic acetylcholine M(2) receptors was examined. The compounds retarded the dissociation of the antagonist [(3)H]N-methylscopolamine ([(3)H]NMS) from porcine cardiac cholinoceptors. This action indicated ternary complex formation. All compounds exhibited higher affinity to the allosteric site of [(3)H]NMS-occupied M(2) receptors than the monomeric strychnine and brucine, while the positive cooperativity with NMS was fully maintained. SAR studies revealed the unchanged strychnine ring as an important structural feature for high allosteric potency.

BDNF Up-Regulates α7 Nicotinic Acetylcholine Receptor Levels on Subpopulations of Hippocampal Interneurons

PubMed Central

Massey, Kerri A.; Zago, Wagner M.; Berg, Darwin K.

2006-01-01

In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of α7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABAA receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased α7-nAChR clusters were most prominent on interneuron subtypes known to innervate directly excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling α7-nAChR levels. PMID:17029981

Muscarinic acetylcholine receptor in cerebellar cortex participates in acetylcholine-mediated blood depressor response in rats.

PubMed

Zhou, Peiling; Zhu, Qingfeng; Liu, Ming; Li, Jing; Wang, Yong; Zhang, Changzheng; Hua, Tianmiao

2015-04-23

Our previous investigations have revealed that cerebellar cholinergic innervation is involved in cardiovascular regulation. This study was performed to examine the effects of the muscarinic cholinergic receptor (mAChR) in the cerebellar cortex on blood pressure (BP) modulation in rats. Acetylcholine (ACh, 100mM), nonselective mAChR agonist (oxotremorine M; Oxo-M, 10, 30 and 100mM) and 100mM ACh mixed with nonselective mAChR antagonist atropine (1, 3 and 10mM) were microinjected into the cerebellar cortex of anesthetized rats. Mean arterial pressure (MAP), maximal decreased MAP (MDMAP), and reaction time (duration required for BP to return to basal values) were measured and analyzed. The results showed that Oxo-M dose-dependently decreased MAP, increased MDMAP, and prolonged reaction time, which displayed a homodromous effect of ACh-mediated blood depressor response; meanwhile, atropine concentration-dependently blocked the effect of ACh on the BP regulation. In conclusion, the present study showed for the first time that mAChRs in cerebellar cortex could modulate somatic BP by participation in ACh-mediated depressor response. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Acetylcholine released from T cells regulates intracellular Ca2+, IL-2 secretion and T cell proliferation through nicotinic acetylcholine receptor.

PubMed

Mashimo, Masato; Iwasaki, Yukari; Inoue, Shoko; Saito, Shoko; Kawashima, Koichiro; Fujii, Takeshi

2017-03-01

T lymphocytes synthesize acetylcholine (ACh) and express muscarinic and nicotinic ACh receptors (mAChR and nAChR, respectively) responsible for increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ). Our aim in the present study was to assess whether autocrine ACh released from T lymphocytes regulates their physiological functions. MOLT-3 human leukemic cell line and murine splenocytes were loaded with fura-2 to monitor [Ca 2+ ] i changes in the absence or presence of several AChR antagonists, including mecamylamine, methyllycaconitine and scopolamine. Real-time PCR and ELISA were performed to measure interleukin-2 (IL-2) mRNA and protein levels. T lymphocytes constitutively produce sufficient amounts of ACh to elicit autocrine changes in [Ca 2+ ] i . These autocrine ACh-evoked [Ca 2+ ] i transients were mediated by nAChRs and then influx of extracellular Ca 2+ . Mecamylamine, a nAChR inhibitor, suppressed not only these [Ca 2+ ] i transients, but also IL-2 release and T cell proliferation. Here, we confirmed that T lymphocytes utilize ACh as a tool to interact with each other and that autocrine ACh-activated nAChRs are involved in cytokine release and cell proliferation. These findings suggest the possibility that nAChR agonists and antagonists and smoking are able to modulate immune function, which in turn suggests the therapeutic potential of immune activation or suppression using nAChR agonists or antagonists. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

PubMed Central

Sniatecki, Jan J.; Goloborodko, Evgeny; Steege, Andreas; Zavaritskaya, Olga; Vetter, Jan M.; Grus, Franz H.; Patzak, Andreas; Wess, Jürgen; Pfeiffer, Norbert

2011-01-01

Purpose. To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. Methods. Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor–deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. Results. Only mRNA for the M3 receptor was detected in retinal arterioles. Thus, M3 receptor–deficient mice (M3R−/−) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R−/− mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R−/−, mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non–subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor Nω-nitro-l-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Conclusions. These findings provide evidence that endothelial M3 receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase. PMID:21873683

Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

PubMed

Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

1994-09-01

Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

Acetylcholine-Like Molecular Arrangement in Psychomimetic Anticholinergic Drugs

PubMed Central

Maayani, Saul; Weinstein, Harel; Cohen, Sasson; Sokolovsky, Mordechai

1973-01-01

A study of the relation between the psychotropic activity and the antagonism to acetylcholine observed for some heterocyclic amino esters and compounds of the phencyclidine series suggests some common molecular structural requirements for their properties. Criteria obtained from quantum mechanical calculations of acetylcholine-like molecules indicate that their molecular reactivity with the cholinergic receptor site follows a certain dynamic interaction pattern. This pattern suggests a certain molecular arrangement essential for the interaction, which is based on the electronic properties of the molecules and therefore remains valid for the evaluation of compounds which lack any apparent similarity to acetylcholine. This type of molecular arrangement is shown to be shared by both activators and inhibitors of the acetylcholine receptor discussed here, thus supporting the hypothesis of their binding to a common receptor. The differences in biological activity are attributed to the effect of molecular structural factors which are not commonly included in the molecular arrangement based on the active groups of acetylcholine. The role of such factors is revealed by a study of the observed differences in the cholinergic and psychomimetic activities of related pairs of isomers and enantiomers of the molecules investigated. Structural factors which interfere with the conformational changes occurring in the receptor protein induced by an activator are characterized through differences obtained by the comparative investigation of the activities of the agonist acetate and the antagonist benzilate amino esters of quinuclidine, tropine, and pseudotropine. The same factors are shown in studies of the phencyclidine series to contribute to the antagonism to acetylcholine activity that is closely related to the psychomimetic activity of these drugs in the central nervous system. Similarly, phencyclidine derivatives in which the characteristic acetylcholine-like molecular

Lead identification of acetylcholinesterase inhibitors-histamine H3 receptor antagonists from molecular modeling.

PubMed

Bembenek, Scott D; Keith, John M; Letavic, Michael A; Apodaca, Richard; Barbier, Ann J; Dvorak, Lisa; Aluisio, Leah; Miller, Kirsten L; Lovenberg, Timothy W; Carruthers, Nicholas I

2008-03-15

Currently, the only clinically effective treatment for Alzheimer's disease (AD) is the use of acetylcholinesterase (AChE) inhibitors. These inhibitors have limited efficacy in that they only treat the symptoms and not the disease itself. Additionally, they often have unpleasant side effects. Here we consider the viability of a single molecule having the actions of both an AChE inhibitor and histamine H(3) receptor antagonist. Both histamine H(3) receptor antagonists and AChE inhibitors improve and augment cholinergic neurotransmission in the cortex. However, whereas an AChE inhibitor will impart its effect everywhere, a histamine H(3) antagonist will raise acetylcholine levels mostly in the brain as its mode of action will primarily be on the central nervous system. Therefore, the combination of both activities in a single molecule could be advantageous. Indeed, studies suggest an appropriate dual-acting compound may offer the desired therapeutic effect with fewer unpleasant side effects [CNS Drugs2004, 18, 827]. Further, recent studies(2) indicate the peripheral anionic site (PAS) of AChE interacts with the beta-amyloid (betaA) peptide. Consequently, a molecule capable of disrupting this interaction may have a significant impact on the production of or the aggregation of betaA. This may result in slowing down the progression of the disease rather than only treating the symptoms as current therapies do. Here, we detail how the use of the available crystal structure information, pharmacophore modeling and docking (automated, manual, classical, and QM/MM) lead to the identification of an AChE inhibitor-histamine H(3) receptor antagonist. Further, based on our models we speculate that this dual-acting compound may interact with the PAS. Such a dual-acting compound may be able to affect the pathology of AD in addition to providing symptomatic relief.

Erysodine, a competitive antagonist at neuronal nicotinic acetylcholine receptors, decreases ethanol consumption in alcohol-preferring UChB rats.

PubMed

Quiroz, Gabriel; Guerra-Díaz, Nicolás; Iturriaga-Vásquez, Patricio; Rivera-Meza, Mario; Quintanilla, María Elena; Sotomayor-Zárate, Ramón

2018-09-03

Alcohol abuse is a worldwide health problem with high economic costs to health systems. Emerging evidence suggests that modulation of brain nicotinic acetylcholine receptors (nAChRs) may be a therapeutic target for alcohol dependence. In this work, we assess the effectiveness of four doses of erysodine (1.5, 2.0, 4.0 or 8.0 mg/kg/day, i.p.), a competitive antagonist of nAChRs, on voluntary ethanol consumption behavior in alcohol-preferring UChB rats, administered during three consecutive days. Results show that erysodine administration produces a dose-dependent reduction in ethanol consumption respect to saline injection (control group). The highest doses of erysodine (4 and 8 mg/kg) reduce (45 and 66%, respectively) the ethanol intake during treatment period and first day of post-treatment compared to control group. While, the lowest doses of erysodine (1.5 and 2 mg/kg) only reduce ethanol intake during one day of treatment period. These effective reductions in ethanol intake were 23 and 29% for 1.5 and 2 mg/kg erysodine, respectively. Locomotor activity induced by a high dose of erysodine (10 mg/kg) was similar to those observed with saline injection in control rats, showing that the reduction in ethanol intake was not produced by hypolocomotor effect induced by erysodine. This is the first report showing that erysodine reduces ethanol intake in UChB rats in a dose-dependent manner. Our results highlight the role of nAChRs in the reward effects of ethanol and its modulation as a potentially effective pharmacological alternative for alcohol dependence treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

NASA Astrophysics Data System (ADS)

Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

2016-03-01

Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

B-973, a novel piperazine positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

PubMed

Post-Munson, Debra J; Pieschl, Rick L; Molski, Thaddeus F; Graef, John D; Hendricson, Adam W; Knox, Ronald J; McDonald, Ivar M; Olson, Richard E; Macor, John E; Weed, Michael R; Bristow, Linda J; Kiss, Laszlo; Ahlijanian, Michael K; Herrington, James

2017-03-15

The alpha7 (α7) nicotinic acetylcholine receptor is a therapeutic target for cognitive disorders. Here we describe 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide (B-973), a novel piperazine-containing molecule that acts as a positive allosteric modulator of the α7 receptor. We characterize the action of B-973 on the α7 receptor using electrophysiology and radioligand binding. At 0.1mM acetylcholine, 1μM B-973 potentiated peak acetylcholine-induced currents 6-fold relative to maximal acetylcholine (3mM) and slowed channel desensitization, resulting in a 6900-fold increase in charge transfer. The EC 50 of B-973 was approximately 0.3μM at acetylcholine concentrations ranging from 0.03 to 3mM. At a concentration of 1μM, B-973 shifted the acetylcholine EC 50 of peak currents from 0.30mM in control to 0.007mM. B-973 slowed channel deactivation upon acetylcholine removal (τ=50s) and increased the affinity of the α7 agonist [ 3 H]A-585539. In the absence of exogenously added acetylcholine, application of B-973 at concentrations >1μM induced large methyllycaconitine-sensitive currents, suggesting B-973 can function as an Ago-PAM at high concentrations. B-973 will be a useful probe for investigating the biological consequences of increasing α7 receptor activity through allosteric modulation. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulation of Neuronal Muscarinic Acetylcholine Receptors

DTIC Science & Technology

1989-01-01

N1E - 115 cells with pertussis toxin blocks mAChR-mediated inhibition of adenylate cyclase but not mAChR-mediated stimulation of PI turnover...determine the effects of electrical depolarization on muscarinic acetylcholine receptors (mAChR) in the cultured neuroblastoma cell line, N E- 115 ...evidence that Gi and Go may differentially regulate cellular signaling mechanisms, these results suggest that depolarization may regulate specific

Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

PubMed

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

2012-12-04

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

Coupling the Torpedo Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins

PubMed Central

Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M.; Zakarian, Armen; Molgó, Jordi

2014-01-01

Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility. PMID:23131021

Acetylcholine activity in selective striatal regions supports behavioral flexibility.

PubMed

Ragozzino, Michael E; Mohler, Eric G; Prior, Margaret; Palencia, Carlos A; Rozman, Suzanne

2009-01-01

Daily living often requires individuals to flexibly respond to new circumstances. There is considerable evidence that the striatum is part of a larger neural network that supports flexible adaptations. Cholinergic interneurons are situated to strongly influence striatal output patterns which may enable flexible adaptations. The present experiments investigated whether acetylcholine actions in different striatal regions support behavioral flexibility by measuring acetylcholine efflux during place reversal learning. Acetylcholine efflux selectively increased in the dorsomedial striatum, but not dorsolateral or ventromedial striatum during place reversal learning. In order to modulate the M2-class of autoreceptors, administration of oxotremorine sesquifumurate (100 nM) into the dorsomedial striatum, concomitantly impaired reversal learning and an increase in acetylcholine output. These effects were reversed by the m(2) muscarinic receptor antagonist, AF-DX-116 (20 nM). The effects of oxotremorine sesquifumurate and AF-DX-116 on acetylcholine efflux were selective to behaviorally-induced changes as neither treatment affected acetylcholine output in a resting condition. In contrast to reversal learning, acetylcholine efflux in the dorsomedial striatum did not change during place acquisition. The results reveal an essential role for cholinergic activity and define its locus of control to the dorsomedial striatum in cognitive flexibility.

Prostanoid receptor antagonists: development strategies and therapeutic applications

PubMed Central

Jones, RL; Giembycz, MA; Woodward, DF

2009-01-01

Identification of the primary products of cyclo-oxygenase (COX)/prostaglandin synthase(s), which occurred between 1958 and 1976, was followed by a classification system for prostanoid receptors (DP, EP1, EP2 …) based mainly on the pharmacological actions of natural and synthetic agonists and a few antagonists. The design of potent selective antagonists was rapid for certain prostanoid receptors (EP1, TP), slow for others (FP, IP) and has yet to be achieved in certain cases (EP2). While some antagonists are structurally related to the natural agonist, most recent compounds are ‘non-prostanoid’ (often acyl-sulphonamides) and have emerged from high-throughput screening of compound libraries, made possible by the development of (functional) assays involving single recombinant prostanoid receptors. Selective antagonists have been crucial to defining the roles of PGD2 (acting on DP1 and DP2 receptors) and PGE2 (on EP1 and EP4 receptors) in various inflammatory conditions; there are clear opportunities for therapeutic intervention. The vast endeavour on TP (thromboxane) antagonists is considered in relation to their limited pharmaceutical success in the cardiovascular area. Correspondingly, the clinical utility of IP (prostacyclin) antagonists is assessed in relation to the cloud hanging over the long-term safety of selective COX-2 inhibitors. Aspirin apart, COX inhibitors broadly suppress all prostanoid pathways, while high selectivity has been a major goal in receptor antagonist development; more targeted therapy may require an intermediate position with defined antagonist selectivity profiles. This review is intended to provide overviews of each antagonist class (including prostamide antagonists), covering major development strategies and current and potential clinical usage. PMID:19624532

Electrophysiological Perspectives on the Therapeutic Use of Nicotinic Acetylcholine Receptor Partial AgonistsS⃞

PubMed Central

Trocmé-Thibierge, Caryn; Guendisch, Daniela; Al Rubaiy, Shehd Abdullah Abbas; Bloom, Stephen A.

2011-01-01

Partial agonist therapies rely variously on two hypotheses: the partial agonists have their effects through chronic low-level receptor activation or the partial agonists work by decreasing the effects of endogenous or exogenous full agonists. The relative significance of these activities probably depends on whether acute or chronic effects are considered. We studied nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes to test a model for the acute interactions between acetylcholine (ACh) and weak partial agonists. Data were best-fit to a basic competition model that included an additional factor for noncompetitive inhibition. Partial agonist effects were compared with the nAChR antagonist bupropion in prolonged bath application experiments that were designed to mimic prolonged drug exposure typical of therapeutic drug delivery. A primary effect of prolonged application of nicotine was to decrease the response of all nAChR subtypes to acute applications of ACh. In addition, nicotine, cytisine, and varenicline produced detectable steady-state activation of α4β2* [(α4)2(β2)3, (α4)3(β2)2, and (α4)2(β2)2α5)] receptor subtypes that was not seen with other test compounds. Partial agonists produced no detectable steady-state activation of α7 nAChR, but seemed to show small potentiation of ACh-evoked responses; however, “run-up” of α7 ACh responses was also sometimes observed under control conditions. Potential off-target effects of the partial agonists therefore included the modulation of α7 responses by α4β2 partial agonists and decreases in α4β2* responses by α7-selective agonists. These data indicate the dual effects expected for α4β2* partial agonists and provide models and insights for utility of partial agonists in therapeutic development. PMID:21285282

Molecular mechanism of acetylcholine receptor-controlled ion translocation across cell membranes

PubMed Central

Cash, Derek J.; Hess, George P.

1980-01-01

Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of 86Rb+ and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1°C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec-1. Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec-1 and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine

Antigenic Structure of the Human Muscle Nicotinic Acetylcholine Receptor Main Immunogenic Region

PubMed Central

Luo, Jie; Lindstrom, Jon

2009-01-01

The main immunogenic region on the α1 subunits of muscle nicotinic acetylcholine receptors provokes half or more of the autoantibodies in myasthenia gravis and its animal model. Many of these autoantibodies depend on the native conformation of the receptor for their ability to bind with high affinity. We mapped this region and explained the conformation-dependence of its epitopes by making chimeras in which sequences of human muscle α1 subunits were replaced in human neuronal α7 subunits or Aplysia acetylcholine binding protein. These chimeras also revealed that the main immunogenic region can play a major role in promoting conformational maturation, and, consequently, assembly of receptor subunits. PMID:19705087

Blockade of central nicotine acetylcholine receptor signaling attenuate ghrelin-induced food intake in rodents.

PubMed

Dickson, S L; Hrabovszky, E; Hansson, C; Jerlhag, E; Alvarez-Crespo, M; Skibicka, K P; Molnar, C S; Liposits, Z; Engel, J A; Egecioglu, E

2010-12-29

Here we sought to determine whether ghrelin's central effects on food intake can be interrupted by nicotine acetylcholine receptor (nAChR) blockade. Ghrelin regulates mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens, partly via cholinergic VTA afferents originating in the laterodorsal tegmental area (LDTg). Given that these cholinergic projections to the VTA have been implicated in natural as well as drug-induced reinforcement, we sought to investigate the role of cholinergic signaling in ghrelin-induced food intake as well as fasting-induced food intake, for which endogenous ghrelin has been implicated. We found that i.p. treatment with the non-selective centrally active nAChR antagonist, mecamylamine decreased fasting-induced food intake in both mice and rats. Moreover, central administration of mecamylamine decreased fasting-induced food intake in rats. I.c.v. ghrelin-induced food intake was suppressed by mecamylamine i.p. but not by hexamethonium i.p., a peripheral nAChR antagonist. Furthermore, mecamylamine i.p. blocked food intake following ghrelin injection into the VTA. Expression of the ghrelin receptor, the growth hormone secretagogue receptor 1A, was found to co-localize with choline acetyltransferase, a marker of cholinergic neurons, in the LDTg. Finally, mecamylamine treatment i.p. decreased the ability of palatable food to condition a place preference. These data suggest that ghrelin-induced food intake is partly mediated via nAChRs and that nicotinic blockade decreases the rewarding properties of food. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

A constitutively active G protein-coupled acetylcholine receptor regulates motility of larval Schistosoma mansoni.

PubMed

MacDonald, Kevin; Kimber, Michael J; Day, Tim A; Ribeiro, Paula

2015-07-01

The neuromuscular system of helminths controls a variety of essential biological processes and therefore represents a good source of novel drug targets. The neuroactive substance, acetylcholine controls movement of Schistosoma mansoni but the mode of action is poorly understood. Here, we present first evidence of a functional G protein-coupled acetylcholine receptor in S. mansoni, which we have named SmGAR. A bioinformatics analysis indicated that SmGAR belongs to a clade of invertebrate GAR-like receptors and is related to vertebrate muscarinic acetylcholine receptors. Functional expression studies in yeast showed that SmGAR is constitutively active but can be further activated by acetylcholine and, to a lesser extent, the cholinergic agonist, carbachol. Anti-cholinergic drugs, atropine and promethazine, were found to have inverse agonist activity towards SmGAR, causing a significant decrease in the receptor's basal activity. An RNAi phenotypic assay revealed that suppression of SmGAR activity in early-stage larval schistosomulae leads to a drastic reduction in larval motility. In sum, our results provide the first molecular evidence that cholinergic GAR-like receptors are present in schistosomes and are required for proper motor control in the larvae. The results further identify SmGAR as a possible candidate for antiparasitic drug targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Altered coupling of muscarinic acetylcholine receptors in pancreatic acinar carcinoma of rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chien, J.L.; Warren, J.R.

The structure and function of muscarinic acetylcholine receptors (mAChR) in acinar carcinoma cells have been compared to mAChR in normal pancreatic acinar cells. Similar 80 kD proteins identified by SDS-PAGE of tumor and normal mAChR affinity-labeled with the muscarinic antagonist /sup 3/H-propylbenzilyl-choline mustards, and identical binding of the antagonist N-methylscopolamine to tumor and normal cells (K/sub D/approx.4x10/sup -10/ M), indicate conservation of mAChR proteins in carcinoma cells. Carcinoma mAChR display homogeneous binding of the agonists carbamylcholine (CCh), K/sub D/approx.3x10/sup -5/ M, and oxotremorine (Oxo), K/sub D/approx.x10/sup -6/ M, whereas normal cells display heterogeneous binding, with a minor component of highmore » affinity interactions for CCh, K/sub D/approx.3x10/sup -6/ M, and Oxo, K/sub D/approx.2x/sup -17/ M, and a major component of low affinity interactions for CCh, K/sub D/approx.1x10/sup -4/ M, and Oxo, K/sub D/approx.2x10/sup -5/ M. Both carcinoma and normal cells exhibit concentration-dependent CCh-stimulated increase in cytosolic free Ca/sup 2 +/, as measured by intracellular Quin 2 fluorescence and /sup 45/Ca/sup 2 +/ efflux. However, carcinoma cells demonstrate 50% maximal stimulation of intracellular Ca/sup 2 +/ release at a CCh concentration (EC/sub 50/approx.6x10/sup -7/ M) one log below that observed for normal cells. The authors propose an altered coupling of mAChR to intracellular Ca/sup 2 +/ homeostasis in carcinoma cells, which is manifest as a single activated receptor state for agonist binding, and increased sensitivity to muscarinic receptor stimulation of Ca/sup 2 +/ release.« less

Agonist-Antagonist Interaction at the Cholinergic Receptor of Denervated Diaphragm,

DTIC Science & Technology

A study has been made of the cholinergic receptor induced by chronic denervation in the rat diaphragm. The agonists acetylcholine, carbachol and...muscle cells. Supramaximally effective doses of agonists caused desensitization of the preparation; however, there was no cross tachyphylaxis between acetylcholine and carbachol . (Author)

Inhibitory effect of strychnine on acetylcholine receptor activation in bovine adrenal medullary chromaffin cells.

PubMed Central

Kuijpers, G A; Vergara, L A; Calvo, S; Yadid, G

1994-01-01

1. Strychnine, which is known as a potent and selective antagonist of the inhibitory glycine receptor in the central nervous system, inhibits the nicotinic stimulation of catecholamine release from bovine cultured adrenal chromaffin cells in a concentration-dependent (1-100 microM) manner. At 10 microM nicotine, the IC50 value for strychnine is approximately 30 microM. Strychnine also inhibits the nicotine-induced membrane depolarization and increase in intracellular Ca2+ concentration. 2. The inhibitory action of strychnine is reversible and is selective for nicotinic stimulation, with no effect observed on secretion elicited by a high external K+ concentration, histamine or angiotensin II. 3. Strychnine competes with nicotine in its effect, but not modify the apparent positive cooperatively of the nicotine binding sites. In the absence of nicotine, strychnine has no effect on catecholamine release. Glycine does not affect catecholamine release nor the inhibitory action of strychnine on this release. 4. These results suggest that strychnine interacts with the agonist binding site of the nicotinic acetylcholine receptor in chromaffin cells, thus exerting a pharmacological effect independently of the glycine receptor. PMID:7834198

Nucleus accumbens acetylcholine and food intake: decreased muscarinic tone reduces feeding but not food-seeking.

PubMed

Pratt, Wayne E; Blackstone, Kaitlin

2009-03-02

Separate groups of food-deprived rats were given 2h access to food after receiving bilateral nucleus accumbens infusions of the muscarinic antagonist scopolamine methyl bromide (at 0, 1.0, and 10.0 microg/side), the M2-preferring agonist oxotremorine sesquifumarate (Oxo-S; at 0, 1.0, or 10.0 microg/side) or the M2 antagonist AFDX-116 (at 0, 0.2, or 1.0 microg/side). Injections of scopolamine or Oxo-S, but not AFDX-116, reduced food consumption across the 2h. These experiments confirm a critical role for Acb acetylcholine in promoting food ingestion, and suggest that decreased acetylcholine tone at post-synaptic muscarinic receptors disrupts normal consummatory behavior.

Synthesis, biological evaluation, and computational studies of Tri- and tetracyclic nitrogen-bridgehead compounds as potent dual-acting AChE inhibitors and hH3 receptor antagonists.

PubMed

Darras, Fouad H; Pockes, Steffen; Huang, Guozheng; Wehle, Sarah; Strasser, Andrea; Wittmann, Hans-Joachim; Nimczick, Martin; Sotriffer, Christoph A; Decker, Michael

2014-03-19

Combination of AChE inhibiting and histamine H3 receptor antagonizing properties in a single molecule might show synergistic effects to improve cognitive deficits in Alzheimer's disease, since both pharmacological actions are able to enhance cholinergic neurotransmission in the cortex. However, whereas AChE inhibitors prevent hydrolysis of acetylcholine also peripherally, histamine H3 antagonists will raise acetylcholine levels mostly in the brain due to predominant occurrence of the receptor in the central nervous system. In this work, we designed and synthesized two novel classes of tri- and tetracyclic nitrogen-bridgehead compounds acting as dual AChE inhibitors and histamine H3 antagonists by combining the nitrogen-bridgehead moiety of novel AChE inhibitors with a second N-basic fragment based on the piperidinylpropoxy pharmacophore with different spacer lengths. Intensive structure-activity relationships (SARs) with regard to both biological targets led to compound 41 which showed balanced affinities as hAChE inhibitor with IC50 = 33.9 nM, and hH3R antagonism with Ki = 76.2 nM with greater than 200-fold selectivity over the other histamine receptor subtypes. Molecular docking studies were performed to explain the potent AChE inhibition of the target compounds and molecular dynamics studies to explain high affinity at the hH3R.

Role of ventrolateral orbital cortex muscarinic and nicotinic receptors in modulation of capsaicin-induced orofacial pain-related behaviors in rats.

PubMed

Tamaddonfard, Esmaeal; Erfanparast, Amir; Abbas Farshid, Amir; Delkhosh-Kasmaie, Fatmeh

2017-11-15

Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5µg/20µl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200ng/site atropine and mecamylamine (200ng/site) prevented oxotremorine (100ng/site)-, and epibatidine (100ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. Copyright © 2017 Elsevier B.V. All rights reserved.

Acetylcholine Activity in Selective Striatal Regions Supports Behavioral Flexibility

PubMed Central

Ragozzino, Michael E.; Mohler, Eric G.; Prior, Margaret; Palencia, Carlos A.; Rozman, Suzanne

2009-01-01

Daily living often requires individuals to flexibly respond to new circumstances. There is considerable evidence that the striatum is part of a larger neural network that supports flexible adaptations. Cholinergic interneurons are situated to strongly influence striatal output patterns which may enable flexible adaptations. The present experiments investigated whether acetylcholine actions in different striatal regions support behavioral flexibility by measuring acetylcholine efflux during place reversal learning. Acetylcholine efflux selectively increased in the dorsomedial striatum, but not dorsolateral or ventromedial striatum during place reversal learning. In order to modulate the M2-class of autoreceptors, administration of oxotremorine sesquifumurate (100 nM) into the dorsomedial striatum, concomitantly impaired reversal learning and an increase in acetylcholine output. These effects were reversed by the m2 muscarinic receptor antagonist, AF-DX-116 (20 nM). The effects of oxotremorine sesquifumurate and AF-DX-116 on acetylcholine efflux were selective to behaviorally-induced changes as neither treatment affected acetylcholine output in a resting condition. In contrast to reversal learning, acetylcholine efflux in the dorsomedial striatum did not change during place acquisition. The results reveal an essential role for cholinergic activity and define its locus of control to the dorsomedial striatum in cognitive flexibility. PMID:18845266

Capsaicin modulates acetylcholine release at the myoneural junction.

PubMed

Thyagarajan, Baskaran; Potian, Joseph G; Baskaran, Padmamalini; McArdle, Joseph J

2014-12-05

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions. Copyright © 2014 Elsevier B.V. All rights reserved.

Desensitization of the nicotinic acetylcholine receptor by diisopropylfluorophosphate.

PubMed

Eldefrawi, M E; Schweizer, G; Bakry, N M; Valdes, J J

1988-01-01

The interaction of diisopropylfluorophosphate (DFP) with the nicotinic acetylcholine (ACh) receptor of Torpedo electric organ was studied, using [3H]-phencyclidine ([3H]-PCP) as a reporter probe. Phencyclidine binds with different kinetics to resting, activated, and desensitized receptor conformations. Although DFP did not inhibit binding of [3H]-ACh or 125I-alpha-bungarotoxin (BGT) to the receptor recognition sites and potentiated in a time-dependent manner [3H]-PCP binding to the receptor's high-affinity allosteric site, it inhibited the ACh- or carbamylcholine-stimulated [3H]-PCP binding. This suggested that DFP bound to a third kind of site on the receptor and affected receptor conformation. Preincubation of the membranes with DFP increased the receptor's affinity for carbamylcholine by eightfold and raised the pseudo-first-order rate of [3H]-PCP binding to that of an agonist-desensitized receptor. Accordingly, it is suggested that DFP induces receptor desensitization by binding to a site that is distinct from the recognition or high-affinity noncompetitive sites.

Galanin antagonizes acetylcholine on a memory task in basal forebrain-lesioned rats.

PubMed

Mastropaolo, J; Nadi, N S; Ostrowski, N L; Crawley, J N

1988-12-01

Galanin coexists with acetylcholine in medial septal neurons projecting to the ventral hippocampus, a projection thought to modulate memory functions. Neurochemical lesions of the nucleus basalis-medial septal area in rats impaired choice accuracy on a delayed alternation t-maze task. Acetylcholine (7.5 or 10 micrograms intraventricularly or 1 micrograms micro-injected into the ventral hippocampus) significantly improved performance in the lesioned rats. Atropine (5 mg/kg intraperitoneally or 10 micrograms intraventricularly), but not mecamylamine (3 mg/kg intraperitoneally or 20 micrograms intraventricularly), blocked this action of acetylcholine, suggesting involvement of a muscarinic receptor. Galanin (100-500 ng intraventricularly or 200 ng into the ventral hippocampus) attenuated the ability of acetylcholine to reverse the deficit in working memory in the lesioned rats. The antagonistic interaction between galanin and acetylcholine suggests that endogenous galanin may inhibit cholinergic function in memory processes, particularly in pathologies such as Alzheimer disease that involve degeneration of basal forebrain neurons.

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

PubMed Central

Armstrong, Lucas C.; Kirsch, Glenn E.; Fedorov, Nikolai B.; Wu, Caiyun; Kuryshev, Yuri A.; Sewell, Abby L.; Liu, Zhiqi; Motter, Arianne L.; Leggett, Carmine S.; Orr, Michael S.

2017-01-01

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors. PMID:28298165

α6β2 nicotinic acetylcholine receptors influence locomotor activity and ethanol consumption.

PubMed

Kamens, Helen M; Peck, Colette; Garrity, Caitlin; Gechlik, Alex; Jenkins, Brenita C; Rajan, Akshat

2017-06-01

Nicotinic acetylcholine receptors (nAChRs) in the mesolimbic dopamine system have been implicated in ethanol behaviors. In particular, work in genetically engineered mice has demonstrated that α6-containing nAChRs are involved in ethanol consumption and sedation. A limitation of these studies is that the alteration in the receptor was present throughout development. The recently described α6β2 antagonist, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), now makes it possible to test for the involvement of these receptors using a pharmacological approach. The aim of this study was to examine the role of α6β2 nAChRs in ethanol behaviors using a pharmacological approach. Adolescent C57BL/6J mice were treated with bPiDI 30 min prior to testing the mice for binge-like ethanol consumption in the drinking-in-the-dark (DID) test, ethanol-induced motor incoordination using the balance beam, and ethanol-induced sedation using the Loss of Righting Reflex (LORR) paradigm. Adolescent animals were chosen because they express a high amount of α6 mRNA relative to adult animals. Control studies were also performed to determine the effect of bPiDI on locomotor activity and ethanol metabolism. Female mice treated with 20 mg/kg bPiDI had reduced locomotor activity compared to saline-treated animals during the first 30 min following an acute injection. Pretreatment with the α6β2 antagonist reduced adolescent ethanol consumption but also reduced saccharin consumption. No significant effects were observed on ethanol-induced ataxia, sedation, or metabolism. This study provides evidence that α6β2 nAChRs are involved in locomotor activity as well as ethanol and saccharin consumption in adolescent animals. Copyright © 2017 Elsevier Inc. All rights reserved.

Metrifonate, like acetylcholine, up-regulates neurotrophic activity of cultured rat astrocytes.

PubMed

Mele, Tina; Jurič, Damijana Mojca

2014-08-01

Metrifonate is an inhibitor of acetylcholinesterase (AChE). Several studies confirmed its positive effects on cognitive impairment in Alzheimer's disease but it was due to adverse events withdrawn from clinical trials. Based on the importance of astrocytes in physiological and pathological brain activities we investigated the impact of metrifonate and, for comparison, acetylcholine on intrinsic neurotrophic activity in these cells. Metabolic activity, intracellular adenosine 5'-triphosphate (ATP) levels and lactate dehydrogenase (LDH) release was measured to examine the impact of metrifonate on viability and integrity of cultured rat cortical astrocytes. The influence of metrifonate, acetylcholine and selective cholinergic ligands on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) synthesis and secretion was determined by specific two-site enzyme immunoassays. Exposure of cultured astrocytes to metrifonate displayed no toxic effects on cell viability. Metrifonate and acetylcholine potently and transiently elevated NGF and BDNF, but not NT-3, protein levels and secretion with different intensity and time frame of their maximal response. Stimulatory effect on NGF was mimicked by selective nicotinic receptor agonist nicotine and completely blocked by nicotinic antagonist mecamylamine. The impact on BDNF synthesis was mimicked by muscarinic receptor agonist pilocarpine and abolished by selective muscarinic antagonist scopolamine. Metrifonate up-regulates astrocytic NGF and BDNF synthesis in the same manner as acetylcholine, their effect depends on different cholinergic pathways. These results suggest a trophic role of metrifonate, based on a well-known neurotrophic activity of NGF and BDNF in vivo. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Effects of neuronal nicotinic acetylcholine receptor allosteric modulators in animal behavior studies

PubMed Central

Pandya, Anshul. A.; Yakel, Jerrel L.

2013-01-01

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation-conducting transmembrane channels from the cys-loop receptor superfamily. The neuronal subtypes of these receptors (e.g. the α7 and α4β2 subtypes) are involved in neurobehavioral processes such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and a number of cognitive functions like learning and memory. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders, and behavioral studies in animals are useful models to assess the effects of compounds that act on these receptors. Allosteric modulators are ligands that bind to the receptors at sites other than the orthosteric site where acetylcholine, the endogenous agonist for the nAChRs, binds. While conventional ligands for the neuronal nAChRs have been studied for their behavioral effects in animals, allosteric modulators for these receptors have only recently gained attention, and research on their behavioral effects is growing rapidly. Here we will discuss the behavioral effects of allosteric modulators of the neuronal nAChRs. PMID:23732296

Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

DOE PAGES

Thal, David M.; Sun, Bingfa; Feng, Dan; ...

2016-03-09

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thal, David M.; Sun, Bingfa; Feng, Dan

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

PubMed

Cambell, B G; Keana, J F; Weber, E

1991-11-26

The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

Modulation of AMPA receptor mediated current by nicotinic acetylcholine receptor in layer I neurons of rat prefrontal cortex

PubMed Central

Tang, Bo; Luo, Dong; Yang, Jie; Xu, Xiao-Yan; Zhu, Bing-Lin; Wang, Xue-Feng; Yan, Zhen; Chen, Guo-Jun

2015-01-01

Layer I neurons in the prefrontal cortex (PFC) exhibit extensive synaptic connections with deep layer neurons, implying their important role in the neural circuit. Study demonstrates that activation of nicotinic acetylcholine receptors (nAChRs) increases excitatory neurotransmission in this layer. Here we found that nicotine selectively increased the amplitude of AMPA receptor (AMPAR)-mediated current and AMPA/NMDA ratio, while without effect on NMDA receptor-mediated current. The augmentation of AMPAR current by nicotine was inhibited by a selective α7-nAChR antagonist methyllycaconitine (MLA) and intracellular calcium chelator BAPTA. In addition, nicotinic effect on mEPSC or paired-pulse ratio was also prevented by MLA. Moreover, an enhanced inward rectification of AMPAR current by nicotine suggested a functional role of calcium permeable and GluA1 containing AMPAR. Consistently, nicotine enhancement of AMPAR current was inhibited by a selective calcium-permeable AMPAR inhibitor IEM-1460. Finally, the intracellular inclusion of synthetic peptide designed to block GluA1 subunit of AMPAR at CAMKII, PKC or PKA phosphorylation site, as well as corresponding kinase inhibitor, blocked nicotinic augmentation of AMPA/NMDA ratio. These results have revealed that nicotine increases AMPAR current by modulating the phosphorylation state of GluA1 which is dependent on α7-nAChR and intracellular calcium. PMID:26370265

Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

NASA Astrophysics Data System (ADS)

Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

2016-08-01

Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

PubMed

Alea, Mileidys Perez; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Fuxe, Kjell; Garriga, Pere

2011-08-15

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function. Copyright © 2011 Elsevier B.V. All rights reserved.

α7 Nicotinic Acetylcholine Receptor Signaling Inhibits Inflammasome Activation by Preventing Mitochondrial DNA Release

PubMed Central

Lu, Ben; Kwan, Kevin; Levine, Yaakov A; Olofsson, Peder S; Yang, Huan; Li, Jianhua; Joshi, Sonia; Wang, Haichao; Andersson, Ulf; Chavan, Sangeeta S; Tracey, Kevin J

2014-01-01

The mammalian immune system and the nervous system coevolved under the influence of cellular and environmental stress. Cellular stress is associated with changes in immunity and activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, a key component of innate immunity. Here we show that α7 nicotinic acetylcholine receptor (α7 nAchR)-signaling inhibits inflammasome activation and prevents release of mitochondrial DNA, an NLRP3 ligand. Cholinergic receptor agonists or vagus nerve stimulation significantly inhibits inflammasome activation, whereas genetic deletion of α7 nAchR significantly enhances inflammasome activation. Acetylcholine accumulates in macrophage cytoplasm after adenosine triphosphate (ATP) stimulation in an α7 nAchR-independent manner. Acetylcholine significantly attenuated calcium or hydrogen oxide–induced mitochondrial damage and mitochondrial DNA release. Together, these findings reveal a novel neurotransmitter-mediated signaling pathway: acetylcholine translocates into the cytoplasm of immune cells during inflammation and inhibits NLRP3 inflammasome activation by preventing mitochondrial DNA release. PMID:24849809

Dopamine D3 receptor antagonism contributes to blonanserin-induced cortical dopamine and acetylcholine efflux and cognitive improvement.

PubMed

Huang, Mei; Kwon, Sunoh; Oyamada, Yoshihiro; Rajagopal, Lakshmi; Miyauchi, Masanori; Meltzer, Herbert Y

2015-11-01

Blonanserin is a novel atypical antipsychotic drug (APD), which, unlike most atypical APDs, has a slightly higher affinity for dopamine (DA) D2 than serotonin (5-HT)2A receptors, and is an antagonist at both, as well as at D3 receptors. The effects of atypical APDs to enhance rodent cortical, hippocampal, limbic, and dorsal striatal (dSTR) DA and acetylcholine (ACh) release, contribute to their ability to improve novel object recognition (NOR) in rodents treated with sub-chronic (sc) phencyclidine (PCP) and cognitive impairment associated with schizophrenia (CIAS). Here we determined the ability of blonanserin, the D3 antagonist NGB 2904, and the typical APD, haloperidol, a D2 antagonist, to enhance neurotransmitter efflux in the medial prefrontal cortex (mPFC) and dSTR of mice, and to ameliorate the scPCP-induced deficit in NOR in rats. Blonanserin, 10mg/kg, i.p., increased DA, norepinephrine (NE), and ACh efflux in mPFC and dSTR. NGB 2904, 3mg/kg, increased DA and ACh, but not NE, efflux in mPFC, and DA, but not ACh, efflux in dSTR. Haloperidol increased DA and NE efflux in dSTR only. The selective D3 agonist PD 128907 partially blocked the blonanserin-induced cortical ACh, DA, NE and striatal DA efflux. NGB 2904, 3mg/kg, like blonanserin, 1mg/kg, and the combination of sub-effective doses of NGB 2904 and blonanserin (both 0.3mg/kg), ameliorated the scPCP-induced NOR deficit in rats. These results suggest that D3 receptor blockade may contribute to the ability of blonanserin to increase cortical DA and ACh efflux, as well as to restore NOR and improve CIAS. Copyright © 2015 Elsevier Inc. All rights reserved.

Activation of alpha-latrotoxin receptors in neuromuscular synapses leads to a prolonged splash acetylcholine release.

PubMed

Lelyanova, V G; Thomson, D; Ribchester, R R; Tonevitsky, E A; Ushkaryov, Y A

2009-06-01

The mechanisms of acetylcholine release in presynaptic terminals of motoneurons induced by mutant alpha-latrotoxin (LT(N4C)) were analyzed. In contrast to wild-type alpha-latrotoxin that causes both continuous and splash secretion of acetylcholine and necessarity block neuromuscular transmission, LT(N4C) causes only splash release lasting over many hours. Thus, activation of alpha-latrotoxin receptors controls long-lasting enhanced secretion of acetylcholine.

Mapping of a binding site for ATP within the extracellular region of the Torpedo nicotinic acetylcholine receptor beta-subunit.

PubMed

Schrattenholz, A; Roth, U; Godovac-Zimmermann, J; Maelicke, A

1997-10-28

Using 2,8,5'-[3H]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant KD of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu11-Pro17 and Asp152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

Agonists and antagonists for P2 receptors

PubMed Central

Jacobson, Kenneth A.; Costanzi, Stefano; Joshi, Bhalchandra V.; Besada, Pedro; Shin, Dae Hong; Ko, Hyojin; Ivanov, Andrei A.; Mamedova, Liaman

2015-01-01

Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X2/3/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X2/3/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformation-ally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4 nM at the P2Y1 receptor, with >10 000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed. PMID:16805423

Lack of CB1 receptors increases noradrenaline release in vas deferens without affecting atrial noradrenaline release or cortical acetylcholine release

PubMed Central

Schlicker, Eberhard; Redmer, Agnes; Werner, André; Kathmann, Markus

2003-01-01

We studied whether cannabinoid CB1 receptor gene disruption (to yield CB1−/− mice) affects the electrically evoked tritium overflow from vas deferens and atrial pieces preincubated with [3H]-noradrenaline (NA) (‘noradrenaline release') and from cerebral cortex slices preincubated with [3H]-choline (‘acetylcholine release'). NA release was higher by 37% in vas deferens from CB1−/− mice than in vas deferens from CB1+/+ mice. The cannabinoid receptor agonist WIN 55,212-2 inhibited, and the CB1 receptor inverse agonist/antagonist SR 141716, increased NA release in vas deferens from CB1+/+ mice without affecting it in vas deferens from CB1−/− mice. Atrial NA release did not differ between CB1+/+ and CB1−/− mice nor did WIN 55,212-2 affect NA release in either strain. Cortical acetylcholine (Ach) release did not differ between CB1+/+ and CB1−/− mice. WIN 55,212-2 inhibited, but SR 141716 did not affect, Ach release in the cortex from CB1+/+ mice. Both drugs did not alter Ach release in the cortex from CB1−/− mice. Tritium content did not differ between CB1+/+ and CB1−/− mice in any preparation. In conclusion, the increase in NA release associated with CB1 receptor deficiency in the vas deferens, which cannot be ascribed to an alteration of tritium content of the preparations, suggests an endogenous tone at the CB1 receptors of CB1+/+ mice in this tissue. Furthermore, the effect of WIN 55,212-2 on NA release in the vas deferens and on cortical Ach release involves CB1 receptors, whereas the involvement of non-CB1–non-CB2 receptors can be excluded. PMID:12970076

Radiosynthesis and evaluation of novel acetylcholine receptor radioligands

NASA Astrophysics Data System (ADS)

Pimlott, Sally L.

Neuroreceptor single photon emission computed tomography (SPECT) imaging provides a powerful tool for the evaluation of the function of a neurotransmitter system in normal and or disease states in the living human brain. The cholinergic system is involved in the control of a variety of complex functions including learning, memory and modulation of behaviour. Deficits in the cholinergic system have been found in a number of neurological diseases, such as Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease and Epilepsy. Acetylcholine receptors (AChRs) are divided into two classes, muscarinic and nicotinic. The aim of this project was to develop two novel SPECT AChR ligands: (R,R)[123I]I-QNB, a M1 subtype selective muscarinic acetylcholine receptor (mAChR) ligand, and 5-[123I]-A-85380, a alpha4beta2 subtype selective nicotinic receptor (nAChR) ligand, for use in human SPECT imaging studies. The calculation of the binding potential of a ligand can be used to obtain quantitative information from a SPECT scan, enabling comparisons to be made between studies. Methodological issues involved in the calculation of binding potential are therefore crucial for the accuracy of results. A particularly important parameter is the amount of authentic radioligand available to cross the blood brain barrier. This was characterised in the research performed for this thesis. The radiosynthesis of two novel neuroreceptor radioligands has been optimised for use in humans. (R, R)[123I]I-QNB has been used in human studies to provide useful information on the human mAChR function in disease. Pre-clinical evaluation of 5-[123I]-A-85380 provided useful information for in vivo human studies. Both radioligands are concluded to successfully provide novel information on the function of the acetylcholine system. Methodological issues involved in the blood metabolite analysis and measurement of plasma protein binding have been investigated and discussed, with particular reference made

Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dwyer, B.P.

1991-04-23

The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digestsmore » of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.« less

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soeda, Junpei; Morgan, Maelle; McKee, Chad

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells inmore » the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms

PubMed Central

Abongwa, Melanie; Buxton, Samuel K.; Robertson, Alan P.; Martin, Richard J.

2016-01-01

Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex

Use of Monoclonal Antibodies to Study the Structure and Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

DTIC Science & Technology

1988-03-16

receptors in muscle is responsible for the muscular weakness characteristic of myasthenia gravis . Some insecticides can act like chemical warfare...expresses muscle-like acetyi-.holine receptor by observing that autoantibodies from myasthenia gravis patients reacted as well with these receptors as...Antibodies in sera from patients with myasthenia gravis do not bind to acetylcholine receptors from human brain. J Neuroimmunol 16:205-213. 21. Whiting

Novel long‐acting antagonists of muscarinic ACh receptors

PubMed Central

Randáková, Alena; Rudajev, Vladimír; Doležal, Vladimír; Boulos, John

2018-01-01

Background and Purpose The aim of this study was to develop potent and long‐acting antagonists of muscarinic ACh receptors. The 4‐hexyloxy and 4‐butyloxy derivatives of 1‐[2‐(4‐oxidobenzoyloxy)ethyl]‐1,2,3,6‐tetrahydropyridin‐1‐ium were synthesized and tested for biological activity. Antagonists with long‐residence time at receptors are therapeutic targets for the treatment of several neurological and psychiatric human diseases. Their long‐acting effects allow for reduced daily doses and adverse effects. Experimental Approach The binding and antagonism of functional responses to the agonist carbachol mediated by 4‐hexyloxy compounds were investigated in CHO cells expressing individual subtypes of muscarinic receptors and compared with 4‐butyloxy analogues. Key Results The 4‐hexyloxy derivatives were found to bind muscarinic receptors with micromolar affinity and antagonized the functional response to carbachol with a potency ranging from 30 nM at M1 to 4 μM at M3 receptors. Under washing conditions to reverse antagonism, the half‐life of their antagonistic action ranged from 1.7 h at M2 to 5 h at M5 receptors. Conclusions and Implications The 4‐hexyloxy derivatives were found to be potent long‐acting M1‐preferring antagonists. In view of current literature, M1‐selective antagonists may have therapeutic potential for striatal cholinergic dystonia, delaying epileptic seizure after organophosphate intoxication or relieving depression. These compounds may also serve as a tool for research into cognitive deficits. PMID:29498041

3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4β2 neuronal nicotinic acetylcholine receptors.

PubMed

Henderson, Brandon J; Orac, Crina M; Maciagiewicz, Iwona; Bergmeier, Stephen C; McKay, Dennis B

2012-02-15

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs. Copyright © 2011 Elsevier Ltd. All rights reserved.

3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4β2 neuronal nicotinic acetylcholine receptors

PubMed Central

Henderson, Brandon J.; Orac, Crina M.; Maciagiewicz, Iwona; Bergmeier, Stephen C.; McKay, Dennis B.

2011-01-01

Subtype selective molecules for α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4β2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3β4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4β2 nAChRs and human α3β4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4β2 nAChRs. PMID:22285942

Strychnine, but not PMBA, inhibits neuronal nicotinic acetylcholine receptors expressed by rabbit retinal ganglion cells.

PubMed

Renna, J M; Strang, C E; Amthor, F R; Keyser, K T

2007-01-01

Strychnine is considered a selective competitive antagonist of glycine gated Cl- channels (Saitoh et al., 1994) and studies have used strychnine at low micromolar concentrations to study the role of glycine in rabbit retina (Linn, 1998; Protti et al., 2005). However, other studies have shown that strychnine, in the concentrations commonly used, is also a potent competitive antagonist of alpha7 nicotinic acetylcholine receptors (nAChRs; Matsubayashi et al., 1998). We tested the effects of low micromolar concentrations of strychnine and 3-[2'-phosphonomethyl[1,1'-biphenyl]-3-yl] alanine (PMBA), a specific glycine receptor blocker (Saitoh et al., 1994; Hosie et al., 1999) on the activation of both alpha7 nAChRs on retinal ganglion cells and on ganglion cell responses to a light flash. Extracellular recordings were obtained from ganglion cells in an isolated retina/choroid preparation and 500 microM choline was used as an alpha7 agonist (Alkondon et al., 1997). We recorded from brisk sustained and brisk transient OFF cells, many of which have been previously shown to have alpha7 receptors (Strang et al., 2005). Further, we tested the effect of strychnine, PMBA and alpha-bungarotoxin on the binding of tetramethylrhodamine alpha-bungarotoxin in the inner plexiform layer. Our data indicates that strychnine, at doses as low as 1.0 microM, can inhibit the alpha7 nAChR-mediated response to choline, but PMBA at concentrations as high as 0.4 microM does not. Binding studies show strychnine and alpha-bungarotoxin inhibit binding of labeled alpha-bungarotoxin in the IPL. Thus, the effects of strychnine application may be to inhibit glycine receptors expressed by ganglion cell or to inhibit amacrine cell alpha7 nAChRs, both of which would result in an increase in the ganglion cell responses. Further research will be required to disentangle the effects of strychnine previously believed to be caused by a single mechanism of glycine receptor inhibition.

Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

NASA Technical Reports Server (NTRS)

Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

1987-01-01

A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

Vasopressin and a nonpeptide antidiuretic hormone receptor antagonist (OPC-31260).

PubMed

Burrell, L M; Phillips, P A; Stephenson, J M; Risvanis, J; Johnston, C I

1994-03-01

The development of nonpeptide orally active AVP analogues has provided a new tool with which to assess the physiological and pathophysiological role of vasopressin (AVP). We have previously characterised the nonpeptide vasopressin V1 receptor antagonist OPC-21268, and now report the in vitro characterisation of the nonpeptide V2 receptor antagonist OPC-31260 in the rat. OPC-31260 caused a concentration-dependent displacement of the selective AVP V2 receptor antagonist radioligand, [3H]desGly-NH2(9)[d(CH2)5, D-Ile2,Ile4]AVP from V2 receptors in rat kidney medulla membranes. The concentration of OPC-31260 that displaced 50% of specific AVP binding (IC50) was 20 +/- 2 nmol/l for renal V2 receptors. OPC-31260 also caused a concentration-dependent displacement of the selective AVP V1 receptor antagonist radioligand, [125I]-[d(CH2)5,sarcosine7]AVP from V1 receptors in both rat liver and kidney medulla membranes. The IC50 was 500 +/- 30 nmol/l for both renal and liver V1 receptors. After oral administration to rats, OPC-31260 was an effective inhibitor of AVP at renal V2 and liver V1 receptors in a time-dependent manner. In vitro binding kinetic studies showed that OPC-31260 was a competitive antagonist at both the renal V2 receptor and the hepatic V1 receptor. OPC-31260 is a nonpeptide, orally effective competitive inhibitor of AVP with a V2:V1 receptor selectivity ratio of 25:1 indicating relative V2 receptor selectivity.

Rapid Antidepressant Actions of Scopolamine: Role of Medial Prefrontal Cortex and M1-subtype Muscarinic Acetylcholine Receptors

PubMed Central

Navarria, Andrea; Wohleb, Eric S.; Voleti, Bhavya; Ota, Kristie T.; Dutheil, Sophie; Lepack, Ashley E.; Dwyer, Jason M.; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S.

2015-01-01

Clinical studies demonstrate that scopolamine, a nonselective muscarinic acetycholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1) in the medial prefrontal cortex (mPFC). The present study extends these findings to determine the role of the mPFC and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. Administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that systemic administration of a selective M1-AChR antagonist, VU0255035 produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions, and found that scopolamine or VU0255035 administration blocked the anhedonic response caused by CUS, an effect that requires chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine, and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. PMID:26102021

Effects of an orally active vasopressin V1 receptor antagonist.

PubMed

Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I

1993-05-01

1. This paper reports on the in vitro and in vivo characteristics of a non-peptide vasopressin V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2( 1H)- quinolinone (OPC-21268). 2. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, [125I]-[d(CH2)5, sarcosine7]AVP from vasopressin V1 receptors in rat liver and kidney membranes, inhibitory concentration of 50% (IC50) 4 x 10(-8), 0.3 mol/L liver and 1.5 x 10(-8), 0.2 mol/L kidney. OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)-d(CH2)5[D-Ileu2, Ileu4]AVP binding to V2 receptors in renal membranes (IC50 > 10(-4) mol/L). 3. After oral administration to rats, OPC-21268 was an effective V1 antagonist to both liver and kidney V1 receptors, in a dose-dependent manner. 4. These studies confirm that OPC-21268 is a potent non-peptide, orally effective V1 vasopressin receptor antagonist.

INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.

EPA Science Inventory

INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.
A.S. Bale*; P.J. Bushnell; C.A. Meacham; T.J. Shafer
Neurotoxicology Division, NHEERL, ORD, US Environmental Protection Agency, Research Triangle Park, NC, USA
Toluene (TOL...

Distinctive Modulation of Dopamine Release in the Nucleus Accumbens Shell Mediated by Dopamine and Acetylcholine Receptors.

PubMed

Shin, Jung Hoon; Adrover, Martin F; Alvarez, Veronica A

2017-11-15

Nucleus accumbens (NAc) shell shows unique dopamine (DA) signals in vivo and plays a unique role in DA-dependent behaviors such as reward-motivated learning and the response to drugs of abuse. A disynaptic mechanism for DA release was reported and shown to require synchronized firing of cholinergic interneurons (CINs) and activation of nicotinic acetylcholine (ACh) receptors (nAChRs) in DA neuron (DAN) axons. The properties of this disynaptic mechanism of DA transmission are not well understood in the NAc shell. In this study, in vitro fast-scan cyclic voltammetry was used to examine the modulation of DA transmission evoked by CINs firing in the shell of mice and compared with other striatal regions. We found that DA signals in the shell displayed significant degree of summation in response to train stimulation of CINs, contrary to core and dorsal striatum. The summation was amplified by a D2-like receptor antagonist and experiments with mice with targeted deletion of D2 receptors to DANs or CINs revealed that D2 receptors in CINs mediate a fast inhibition observed within 100 ms of the first pulse, whereas D2 autoreceptors in DAN terminals are engaged in a slower inhibition that peaks at ∼500 ms. ACh also contributes to the use-dependent inhibition of DA release through muscarinic receptors only in the shell, where higher activity of acetylcholinesterase minimizes nAChR desensitization and promotes summation. These findings show that DA signals are modulated differentially by endogenous DA and ACh in the shell, which may underlie the unique features of shell DA signals in vivo SIGNIFICANCE STATEMENT The present study reports that dopamine (DA) release evoked by activation of cholinergic interneurons displays a high degree of summation in the shell and shows unique modulation by endogenous DA and acetylcholine. Desensitization of nicotinic receptors, which is a prevailing mechanism for use-dependent inhibition in the nucleus accumbens core and dorsal striatum, is

Structure and dynamics of the M3 muscarinic acetylcholine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.

2012-03-01

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify themore » binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.« less

Thinking in cycles: MWC is a good model for acetylcholine receptor-channels

PubMed Central

Auerbach, Anthony

2012-01-01

Abstract Neuromuscular acetylcholine receptors have long been a model system for understanding the mechanisms of operation of ligand-gated ion channels and fast chemical synapses. These five subunit membrane proteins have two allosteric (transmitter) binding sites and a distant ion channel domain. Occupation of the binding sites by agonist molecules transiently increases the probability that the channel is ion-permeable. Recent experiments show that the Monod, Wyman and Changeux formalism for allosteric proteins, originally developed for haemoglobin, is an excellent model for acetylcholine receptors. By using mutations and single-channel electrophysiology, the gating equilibrium constants for receptors with zero, one or two bound agonist molecules, and the agonist association and dissociation rate constants from both the closed- and open-channel conformations, have been estimated experimentally. The change in affinity for each transmitter molecule between closed and open conformations provides ∼–5.1 kcal mol−1 towards the global gating isomerization of the protein. PMID:21807612

Implementation of a Fluorescence-Based Screening Assay Identifies Histamine H3 Receptor Antagonists Clobenpropit and Iodophenpropit as Subunit-Selective N-Methyl-d-Aspartate Receptor Antagonists

PubMed Central

Hansen, Kasper B.; Mullasseril, Praseeda; Dawit, Sara; Kurtkaya, Natalie L.; Yuan, Hongjie; Vance, Katie M.; Orr, Anna G.; Kvist, Trine; Ogden, Kevin K.; Le, Phuong; Vellano, Kimberly M.; Lewis, Iestyn; Kurtkaya, Serdar; Du, Yuhong; Qui, Min; Murphy, T. J.; Snyder, James P.; Bräuner-Osborne, Hans

2010-01-01

N-Methyl-d-aspartate (NMDA) receptors are ligand-gated ion channels that mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the central nervous system and play a pivotal role in synaptic plasticity, neuronal development, and several neurological diseases. We describe a fluorescence-based assay that measures NMDA receptor-mediated changes in intracellular calcium in a BHK-21 cell line stably expressing NMDA receptor NR2D with NR1 under the control of a tetracycline-inducible promoter (Tet-On). The assay selectively identifies allosteric modulators by using supramaximal concentrations of glutamate and glycine to minimize detection of competitive antagonists. The assay is validated by successfully identifying known noncompetitive, but not competitive NMDA receptor antagonists among 1800 screened compounds from two small focused libraries, including the commercially available library of pharmacologically active compounds. Hits from the primary screen are validated through a secondary screen that used two-electrode voltage-clamp recordings on recombinant NMDA receptors expressed in Xenopus laevis oocytes. This strategy identified several novel modulators of NMDA receptor function, including the histamine H3 receptor antagonists clobenpropit and iodophenpropit, as well as the vanilloid receptor transient receptor potential cation channel, subfamily V, member 1 (TRPV1) antagonist capsazepine. These compounds are noncompetitive antagonists and the histamine H3 receptor ligand showed submicromolar potency at NR1/NR2B NMDA receptors, which raises the possibility that compounds can be developed that act with high potency on both glutamate and histamine receptor systems simultaneously. Furthermore, it is possible that some actions attributed to histamine H3 receptor inhibition in vivo may also involve NMDA receptor antagonism. PMID:20197375

Characterization of Ganglionic Acetylcholine Receptor Autoantibodies

PubMed Central

Vernino, Steven; Lindstrom, Jon; Hopkins, Steve; Wang, Zhengbei; Low, Phillip A.

2008-01-01

In myasthenia gravis (MG), autoantibodies bind to the α1 subunit and other subunits of the muscle nicotinic acetylcholine receptor (AChR). Autoimmune autonomic ganglionopathy (AAG) is an antibody-mediated neurological disorder caused by antibodies against neuronal AChRs in autonomic ganglia. Subunits of muscle and neuronal AChR are homologous. We examined the specificity of AChR antibodies in patients with MG and AAG. Ganglionic AChR autoantibodies found in AAG patients are specific for AChRs containing the α3 subunit. Muscle and ganglionic AChR antibody specificities are distinct. Antibody crossreactivity between AChRs with different α subunits is uncommon but can occur. PMID:18485491

NOP Receptor Mediates Anti-analgesia Induced by Agonist-Antagonist Opioids

PubMed Central

Gear, Robert W.; Bogen, Oliver; Ferrari, Luiz F.; Green, Paul G.; Levine, Jon D.

2014-01-01

Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ~90 minutes after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69,593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

Acetylcholine Attenuates Hydrogen Peroxide-Induced Intracellular Calcium Dyshomeostasis Through Both Muscarinic and Nicotinic Receptors in Cardiomyocytes.

PubMed

Palee, Siripong; Apaijai, Nattayaporn; Shinlapawittayatorn, Krekwit; Chattipakorn, Siriporn C; Chattipakorn, Nipon

2016-01-01

Oxidative stress induced intracellular Ca2+ overload plays an important role in the pathophysiology of several heart diseases. Acetylcholine (ACh) has been shown to suppress reactive oxygen species generation during oxidative stress. However, there is little information regarding the effects of ACh on the intracellular Ca2+ regulation in the presence of oxidative stress. Therefore, we investigated the effects of ACh applied before or after hydrogen peroxide (H2O2) treatment on the intracellular Ca2+ regulation in isolated cardiomyocytes. Single ventricular myocytes were isolated from the male Wistar rats for the intracellular Ca2+ transient study by a fluorimetric ratio technique. H2O2 significantly decreased both of intracellular Ca2+ transient amplitude and decay rate. ACh applied before, but not after, H2O2 treatment attenuated the reduction of intracellular Ca2+ transient amplitude and decay rate. Both atropine (a muscarinic acetylcholine receptor blocker) and mecamylamine (a nicotinic acetylcholine receptor blocker) significantly decreased the protective effects of acetylcholine on the intracellular Ca2+ regulation. Moreover, the combination of atropine and mecamylamine completely abolished the protective effects of acetylcholine on intracellular Ca2+ transient amplitude and decay rate. ACh pretreatment attenuates H2O2-induced intracellular Ca2+ dyshomeostasis through both muscarinic and nicotinic receptors. © 2016 The Author(s) Published by S. Karger AG, Basel.

Nucleus accumbens neurotransmission and effort-related choice behavior in food motivation: effects of drugs acting on dopamine, adenosine, and muscarinic acetylcholine receptors.

PubMed

Nunes, Eric J; Randall, Patrick A; Podurgiel, Samantha; Correa, Mercè; Salamone, John D

2013-11-01

Mesolimbic dopamine (DA) is a critical component of the brain circuitry regulating behavioral activation and effort-related processes. Although nucleus accumbens (NAc) DA depletions or antagonism leave aspects of appetite and primary food motivation intact, rats with impaired DA transmission reallocate their instrumental behavior away from food-reinforced tasks with high response requirements, and instead select less effortful food-seeking behaviors. Previous work showed that adenosine A2A antagonists can reverse the effects of DA D2 antagonists on effort-related choice, and that stimulation of adenosine A2A receptors produces behavioral effects that are similar to those induced by DA antagonism. The present review summarizes the literature on the role of NAc DA and adenosine in effort-related processes, and also presents original data on the effects of local stimulation of muscarinic acetylcholine receptors in NAc core. Local injections of the muscarinic agonist pilocarpine directly into NAc core produces shifts in effort-related choice behavior similar to those induced by DA antagonism or A2A receptor stimulation, decreasing lever pressing but increasing chow intake in rats responding on a concurrent fixed ratio/chow feeding choice task. In contrast, injections into a neostriatal control site dorsal to the NAc were ineffective. The actions of pilocarpine on this task were attenuated by co-administration of the muscarinic antagonist scopolamine. Thus, drugs that act on DA, adenosine A2A, and muscarinic receptors regulate effort-related choice behavior, which may have implications for the treatment of psychiatric symptoms such as psychomotor slowing, fatigue or anergia that can be observed in depression and other disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

PubMed

Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

2017-01-18

Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

αConotoxin ArIB[V11L,V16D] is a potent and selective antagonist at rat and human native α7 nicotinic acetylcholine receptors

PubMed Central

Innocent, Neal; Livingstone, Phil D.; Hone, Arik; Kimura, Atsuko; Young, Tracey; Whiteaker, Paul; McIntosh, J. Michael; Wonnacott, Susan

2008-01-01

A recently developed α-conotoxin, α-CtxArIB[V11L,V16D] is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues where they are implicated in a variety of functions. Here we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 in rat PC12 cells and human SHSY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli (5-iodo-A-85380, nicotine or KCl) that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]: ACh-evoked currents in X. laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the timecourse of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces: α-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions. PMID:18664588

Muscarinic acetylcholine receptor activation blocks long-term potentiation at cerebellar parallel fiber-Purkinje cell synapses via cannabinoid signaling.

PubMed

Rinaldo, Lorenzo; Hansel, Christian

2013-07-02

Muscarinic acetylcholine receptors (mAChRs) are known to modulate synaptic plasticity in various brain areas. A signaling pathway triggered by mAChR activation is the production and release of endocannabinoids that bind to type 1 cannabinoid receptors (CB1R) located on synaptic terminals. Using whole-cell patch-clamp recordings from rat cerebellar slices, we have demonstrated that the muscarinic agonist oxotremorine-m (oxo-m) blocks the induction of presynaptic long-term potentiation (LTP) at parallel fiber (PF)-Purkinje cell synapses in a CB1R-dependent manner. Under control conditions, LTP was induced by delivering 120 PF stimuli at 8 Hz. In contrast, no LTP was observed when oxo-m was present during tetanization. PF-LTP was restored when the CB1R antagonist N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM251) was coapplied with oxo-m. Furthermore, the suppressive effect of oxo-m on PF-LTP was abrogated by the GDP analog GDP-β-S (applied intracellularly), the phospholipase C inhibitor U-73122, and the diacylglycerol lipase inhibitor tetrahydrolipstatin (THL), suggesting that cannabinoid synthesis results from the activation of Gq-coupled mAChRs present on Purkinje cells. The oxo-m-mediated suppression of LTP was also prevented in the presence of the M3 receptor antagonist DAU 5884, and was absent in M1/M3 receptor double-KO mice, identifying M3 receptors as primary oxo-m targets. Our findings allow for the possibility that cholinergic signaling in the cerebellum--which may result from long-term depression (LTD)-related disinhibition of cholinergic neurons in the vestibular nuclei--suppresses presynaptic LTP to prevent an up-regulation of transmitter release that opposes the reduction of postsynaptic responsiveness. This modulatory capacity of mAChR signaling could promote the functional penetrance of LTD.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

2016-01-01

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes. �

The wonderland of neuronal nicotinic acetylcholine receptors.

PubMed

Bertrand, Daniel; Terry, A V

2018-05-01

Nearly 30 years of experimental evidence supports the argument that ligands of nicotinic acetylcholine receptors (nAChRs) have potential as therapeutic agents. However, as in the famous Lewis Carroll novel "Alice in Wonderland", there have been many unexpected adventures along the pathway of development, and few nAChR ligands have been approved for any clinical condition to date with the exception of nicotine dependence. The recent failures of nAChR ligands in AD and schizophrenia clinical trials have reduced enthusiasm for this therapeutic strategy and many pharmaceutical companies have now abandoned this field of research. As with other clinical failures, multiple questions arise as to the basis for the failure. More generic questions focus on a potential translational gap between the animal models used and the human clinical condition they are meant to simulate, or the clinical trial mindset that large Ns have to be achieved for statistical power (often requiring multiple trial sites) as opposed to smaller patient cohorts at limited sites where conditions can be better controlled and replicated. More specific to the nAChR field are questions about subtype selectivity, dose selection, whether an agonist, antagonist, or allosteric modulator strategy is best, etc. The purpose of this review is to discuss each of these questions, but also to provide a brief overview of the remarkable progress that has been made over the last three decades in our understanding of this unique ligand-gated ion channel and how this new knowledge may help us improve drug development successes in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural Modifications to Tetrahydropyridine-3-Carboxylate Esters en route to the Discovery of M5-Preferring Muscarinic Receptor Orthosteric Antagonists

PubMed Central

Zheng, Guangrong; Smith, Andrew M.; Huang, Xiaoqin; Subramanian, Karunai L.; Siripurapu, Kiran B.; Deaciuc, Agripina; Zhan, Chang-Guo; Dwoskin, Linda P.

2013-01-01

The M5 muscarinic acetylcholine receptor is suggested to be a potential pharmacotherapeutic target for the treatment of drug abuse. We describe herein the discovery of a series of M5-preferring orthosteric antagonists based on the scaffold of 1,2,5,6-tetrahydropyridine-3-carboxylic acid. Compound 56, the most selective compound in this series, possesses an 11-fold selectivity for the M5 over M1 receptor, and shows little activity at M2–M4. This compound, although exhibiting modest affinity (Ki = 2.24 μM) for the [3H]N-methylscopolamine binding site on the M5 receptor, is potent (IC50 = 0.45 nM) in inhibiting oxotremorine-evoked [3H]DA release from rat striatal slices. Further, a homology model of human M5 receptor based on the crystal structure of the rat M3 receptor was constructed, and docking studies of compounds 28 and 56 were performed in an attempt to understand the possible binding mode of these novel analogues to the receptor. PMID:23379472

Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

PubMed Central

Freeman, Hugh J; Gillett, Helen R; Gillett, Peter M; Oger, Joel

2009-01-01

Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a gluten-free diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms. PMID:19824105

Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

PubMed

Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

1998-07-24

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

Pharmacological and ionic characterizations of the muscarinic receptors modulating (/sup 3/H)acetylcholine release from rat cortical synaptosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyer, E.M.; Otero, D.H.

The muscarinic receptors that modulate acetylcholine release from rat cortical synaptosomes were characterized with respect to sensitivity to drugs that act selectively at M1 or M2 receptor subtypes, as well as to changes in ionic strength and membrane potential. The modulatory receptors appear to be of the M2 type, since they are activated by carbachol, acetylcholine, methacholine, oxotremorine, and bethanechol, but not by pilocarpine, and are blocked by atropine, scopolamine, and gallamine (at high concentrations), but not by pirenzepine or dicyclomine. The ED50S for carbachol, acetylcholine, and oxotremorine are less than 10 microM, suggesting that the high affinity state ofmore » the receptor is functional. High ionic strength induced by raising the NaCl concentration has no effect on agonist (oxotremorine) potency, but increases the efficacy of this compound, which disagrees with receptor-binding studies. On the other hand, depolarization with either KCl or with veratridine (20 microM) reduces agonist potencies by approximately an order of magnitude, suggesting a potential mechanism for receptor regulation.« less

Kinin B1 receptor antagonists containing alpha-methyl-L-phenylalanine: in vitro and in vivo antagonistic activities.

PubMed

Gobeil, F; Charland, S; Filteau, C; Perron, S I; Neugebauer, W; Regoli, D

1999-03-01

-To protect from metabolism and to improve potency of the AcLys-[D-betaNal7,Ile8]desArg9-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing alpha-methyl-L-Phe ([alphaMe]Phe) in position 5: these are the AcLys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 892), Lys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 913), and AcLys-Lys-[(alphaMe)Phe5,D-betaNal7, Ile8]desArg9BK (R 914). The new compounds were tested against the contractile effect induced by desArg9BK on 2 B1 receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu8]desArg9BK and [Leu8]desArg9BK) and with other recently described peptide antagonists. The 3 (alphaMe)Phe analogues showed high antagonistic potencies (pA2) at both the human (8.8, 7.7, and 8. 7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B1 receptors. No antagonistic effects (pA2<5) were observed on the B2 receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B1 antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The Nalpha-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID50) of B1 receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B1 receptor) and nontreated (for B2 receptor) rabbits against the hypotensive effects of exogenous desArg9BK and BK. R 892 efficiently inhibited (ID50 2.8 nmol/kg IV) hypotension induced by desArg9BK without affecting that evoked by BK (ID50 >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp3,Igl5,D-Igl7,Oic8]desArg9BK (B 9858) and DArg-[Hyp3,Thi5,D-Tic7,Oic8] desArg9BK (S 0765) showed dual B1/B2 receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective

μ Opioid receptor: novel antagonists and structural modeling

NASA Astrophysics Data System (ADS)

Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela

2016-02-01

The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.

Prolonged administration of pyridostigmine impairs neuromuscular function with and without down-regulation of acetylcholine receptors.

PubMed

Richtsfeld, Martina; Yasuhara, Shingo; Fink, Heidrun; Blobner, Manfred; Martyn, J A Jeevendra

2013-08-01

The acetylcholinesterase inhibitor, pyridostigmine, is prophylactically administered to mitigate the toxic effects of nerve gas poisoning. The authors tested the hypothesis that prolonged pyridostigmine administration can lead to neuromuscular dysfunction and even down-regulation of acetylcholine receptors. Pyridostigmine (5 or 25 mg·kg·day) or saline was continuously administered via osmotic pumps to rats, and infused for either 14 or 28 days until the day of neuromuscular assessment (at day 14 or 28), or discontinued 24 h before neuromuscular assessment. Neurotransmission and muscle function were examined by single-twitch, train-of-four stimulation and 100-Hz tetanic stimulation. Sensitivity to atracurium and acetylcholine receptor number (quantitated by I-α-bungarotoxin) provided additional measures of neuromuscular integrity. Specific tetanic tensions (Newton [N]/muscle weight [g]) were significantly (P < 0.05) decreased at 14 (10.3 N/g) and 28 (11.1 N/g) days of 25 mg·kg·day pyridostigmine compared with controls (13.1-13.6 N/g). Decreased effective dose (0.81-1.05 vs. 0.16-0.45 mg/kg; P < 0.05) and decreased plasma concentration (3.02-3.27 vs. 0.45-1.37 μg/ml; P < 0.05) of atracurium for 50% paralysis (controls vs. 25 mg·kg·day pyridostigmine, respectively), irrespective of discontinuation of pyridostigmine, confirmed the pyridostigmine-induced altered neurotransmission. Pyridostigmine (25 mg·kg·day) down-regulated acetylcholine receptors at 28 days. Prolonged administration of pyridostigmine (25 mg·kg·day) leads to neuromuscular impairment, which can persist even when pyridostigmine is discontinued 24 h before assessment of neuromuscular function. Pyridostigmine has the potential to down-regulate acetylcholine receptors, but induces neuromuscular dysfunction even in the absence of receptor changes.

Interactions of agonists with an allosteric antagonist at muscarinic acetylcholine M2 receptors.

PubMed

Lanzafame, A; Christopoulos, A; Mitchelson, F

1996-11-28

The interaction of heptane-1,7-bis(dimethyl-3'-phthalimidopropylammonium bromide) (C7/3'-phth), with several agonists, was investigated at the muscarinic M2 receptor in guinea-pig left atria. C7/3'-phth shifted concentration-response curves for the agonists, carbachol, oxotremorine-M and (+)-cis-dioxolane, to the right in a parallel fashion. Arunlakshana-Schild regressions of the data yielded slopes significantly different to unity, suggesting non-competitive antagonism. Non-linear regression analysis, using an equation based on allosteric modulation, gave quantitative estimates of co-operativity (alpha values) and the dissociation constant of C7/3'-phth (KB). In all cases, the KB estimates for C7/3'-phth were not significantly different. Increasing the carbachol contact time 10-fold did not significantly influence the KB or the alpha value obtained with C7/3'-phth. Changing from Krebs to Tyrode solution did not significantly alter the KB for C7/3'-phth, although alpha values obtained were consistently lower in Tyrode solution, suggesting that the allosteric action may be sensitive to buffer composition. A 4-fold higher degree of negative, heterotropic co-operativity between C7/3'-phth and agonists than between C7/3'-phth and competitive antagonists was also found.

Functional somato-dendritic α7-containing nicotinic acetylcholine receptors in the rat basolateral amygdala complex

PubMed Central

Klein, Rebecca C; Yakel, Jerrel L

2006-01-01

Multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed in the CNS. The amygdala complex, the limbic structure important for emotional memory formation, receives cholinergic innervation from the basal forebrain. Although cholinergic drugs have been shown to regulate passive avoidance performance via the amygdala, the neuronal subtypes and circuits involved in this regulation are unknown. In the present study, whole-cell patch-clamp electrophysiological techniques were used to identify and characterize the presence of functional somato-dendritic nAChRs within the basolateral complex of the amygdala. Pressure-application of acetylcholine (ACh; 2 mm) evoked inward current responses in a subset of neurons from both the lateral (49%) and basolateral nuclei (72%). All responses displayed rapid activation kinetics, and were blocked by the α7-selective antagonist methyllycaconitine. In addition, the α7-selective agonist choline induced inward current responses that were similar to ACh-evoked responses. Spiking patterns were consistent with pyramidal class I neurons (the major neuronal type in the basolateral complex); however, there was no correlation between firing frequency and the response to ACh. The local photolysis of caged carbachol demonstrated that the functional expression of nAChRs is located both on the soma and dendrites. This is the first report demonstrating the presence of functional nAChR-mediated current responses from rat amygdala slices, where they may be playing a significant role in fear and aversively motivated memory. PMID:16931547

Nicotinic Acetylcholine Receptors Mediate the Suppressive Effect of an Injection of Diluted Bee Venom into the GV3 Acupoint on Oxaliplatin-Induced Neuropathic Cold Allodynia in Rats.

PubMed

Yoon, Heera; Kim, Min Joon; Yoon, Insoo; Li, Dong Xing; Bae, Hyunsu; Kim, Sun Kwang

2015-01-01

Oxaliplatin, a platinum-based chemotherapy drug, often induces acute neuropathic pain, especially cold allodynia, even after a single administration. Subcutaneous injection of diluted bee venom (BV) into acupoints has been used to treat various pain symptoms in traditional oriental medicine. Although we previously demonstrated the suppressive effect of BV injection on oxaliplatin-induced cold allodynia in rats, its neurochemical mechanism remained unclear. This study investigates whether and how the cholinergic system mediates the relieving effect of BV injection on cold allodynia in oxaliplatin-administered rats. The behavioral signs of cold allodynia induced by an oxaliplatin administration (6 mg/kg, intraperitoneally (i.p.)) were evaluated by a tail immersion test in cold water (4°C). BV (0.25 mg/kg, subcutaneously (s.c.)) injection into the Yaoyangguan acupoint, located between the spinous processes of the fourth and fifth lumbar vertebrae, significantly alleviated the cold allodynia. This relieving effect of BV injection on oxaliplatin-induced cold allodynia was blocked by a pretreatment with mecamylamine (a non-selective nicotinic receptor antagonist, 2 mg/kg, i.p.), but not by atropine (a non-selective muscarinic receptor antagonist, 1 mg/kg, i.p.). Further, dihydro-β-erythroidinehydrobromide (DHβE, an α4β2 nicotinic antagonist, 5 mg/kg, i.p.) prevented the anti-allodynic effect of BV, whereas methyllycaconitine (an α7 nicotinic antagonist, 6 mg/kg, i.p.) did not. Finally, intrathecal administration of DHβE (10 nM) blocked the BV-induced anti-allodynic effect. These results suggest that nicotinic acetylcholine receptors, especially spinal α4β2 receptors, but not muscarinic receptors, mediate the suppressive effect of BV injection on oxaliplatin-induced acute cold allodynia in rats.

Non-selectivity of new bradykinin antagonists for B1 receptors.

PubMed

Rhaleb, N E; Gobeil, F; Regoli, D

1992-01-01

Two new B1 receptor antagonists, [Hyp3,Thi5,DTic7,Oic8]desArg9-BK and DArg[Hyp3,Thi5,DTic7,Oic8]desArg9-BK were tested in vitro on the rabbit jugular vein and the guinea pig ileum (preparations containing B2 receptors) and on the rabbit aorta (preparation containing B1 receptors) for pharmacological characterization. The results indicate that both compounds are antagonists on both B1 and B2 receptors, are competitive and discriminate between B2A and B2B receptor subtypes.

In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist.

PubMed

Emonds-Alt, X; Doutremepuich, J D; Heaulme, M; Neliat, G; Santucci, V; Steinberg, R; Vilain, P; Bichon, D; Ducoux, J P; Proietto, V

1993-12-21

(S)1-(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pip eridin-3- yl]ethyl)-4-phenyl-1-azoniabicyclo[2.2.2]octane chloride (SR140333) is a new non-peptide antagonist of tachykinin NK1 receptors. SR140333 potently, selectively and competitively inhibited substance P binding to NK1 receptors from various animal species, including humans. In vitro, it was a potent antagonist in functional assays for NK1 receptors such as [Sar9,Met(O2)11]substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery and contraction of guinea-pig ileum. Up to 1 microM, it had no effect in bioassays for NK2 ([beta Ala8]neurokinin A-induced contraction of endothelium-deprived rabbit pulmonary artery) and NK3 ([MePhe7]neurokinin B-induced contraction of rat portal vein) receptors. The antagonism exerted by SR140333 toward NK1 receptors was apparently non-competitive, with pD2' values (antagonism potency evaluated by the negative logarithm of the molar concentration of antagonist that produces a 50% reduction of the maximal response to the agonist) between 9.65 and 10.16 in the different assays. SR140333 also blocked in vitro [Sar9,Met(O2)11]substance P-induced release of acetylcholine from rat striatum. In vivo, SR140333 exerted highly potent antagonism toward [Sar9,Met(O2)11]substance P-induced hypotension in dogs (ED50 = 3 micrograms/kg i.v.), bronchoconstriction in guinea-pig (ED50 = 42 micrograms/kg i.v.) and plasma extravasation in rats (ED50 = 7 micrograms/kg i.v.). Finally, it also blocked the activation of rat thalamic neurons after nociceptive stimulation (ED50 = 0.2 micrograms/kg i.v.).

Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.

PubMed

Lefebvre, Romain A; Van Colen, Inge; Pauwelyn, Vicky; De Maeyer, Joris H

2016-06-15

5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [(3)H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1-1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01-0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Muscarinic Acetylcholine Receptors Act in Synergy to Facilitate Learning and Memory

ERIC Educational Resources Information Center

Leaderbrand, Katherine; Chen, Helen J.; Corcoran, Kevin A.; Guedea, Anita L.; Jovasevic, Vladimir; Wess, Jurgen; Radulovic, Jelena

2016-01-01

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on…

Adenosine A₁ and A₂A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction.

PubMed

Garcia, Neus; Priego, Mercedes; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Besalduch, Nuria; Lanuza, M Angel; Tomàs, Josep

2013-07-01

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

PubMed Central

Ajram, Laura; Begg, Malcolm; Slack, Robert; Cryan, Jenni; Hall, David; Hodgson, Simon; Ford, Alison; Barnes, Ashley; Swieboda, Dawid; Mousnier, Aurelie; Solari, Roberto

2014-01-01

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target. PMID:24534492

Acetylcholine modulates gamma frequency oscillations in the hippocampus by activation of muscarinic M1 receptors.

PubMed

Betterton, Ruth T; Broad, Lisa M; Tsaneva-Atanasova, Krasimira; Mellor, Jack R

2017-06-01

Modulation of gamma oscillations is important for the processing of information and the disruption of gamma oscillations is a prominent feature of schizophrenia and Alzheimer's disease. Gamma oscillations are generated by the interaction of excitatory and inhibitory neurons where their precise frequency and amplitude are controlled by the balance of excitation and inhibition. Acetylcholine enhances the intrinsic excitability of pyramidal neurons and suppresses both excitatory and inhibitory synaptic transmission, but the net modulatory effect on gamma oscillations is not known. Here, we find that the power, but not frequency, of optogenetically induced gamma oscillations in the CA3 region of mouse hippocampal slices is enhanced by low concentrations of the broad-spectrum cholinergic agonist carbachol but reduced at higher concentrations. This bidirectional modulation of gamma oscillations is replicated within a mathematical model by neuronal depolarisation, but not by reducing synaptic conductances, mimicking the effects of muscarinic M1 receptor activation. The predicted role for M1 receptors was supported experimentally; bidirectional modulation of gamma oscillations by acetylcholine was replicated by a selective M1 receptor agonist and prevented by genetic deletion of M1 receptors. These results reveal that acetylcholine release in CA3 of the hippocampus modulates gamma oscillation power but not frequency in a bidirectional and dose-dependent manner by acting primarily through muscarinic M1 receptors. © 2017 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Orexin OX2 Receptor Antagonists as Sleep Aids.

PubMed

Jacobson, Laura H; Chen, Sui; Mir, Sanjida; Hoyer, Daniel

The discovery of the orexin system represents the single major progress in the sleep field of the last three to four decades. The two orexin peptides and their two receptors play a major role in arousal and sleep/wake cycles. Defects in the orexin system lead to narcolepsy with cataplexy in humans and dogs and can be experimentally reproduced in rodents. At least six orexin receptor antagonists have reached Phase II or Phase III clinical trials in insomnia, five of which are dual orexin receptor antagonists (DORAs) that target both OX 1 and OX 2 receptors (OX 2 Rs). All clinically tested DORAs induce and maintain sleep: suvorexant, recently registered in the USA and Japan for insomnia, represents the first hypnotic principle that acts in a completely different manner from the current standard medications. It is clear, however, that in the clinic, all DORAs promote sleep primarily by increasing rapid eye movement (REM) and are almost devoid of effects on slow-wave (SWS) sleep. At present, there is no consensus on whether the sole promotion of REM sleep has a negative impact in patients suffering from insomnia. However, sleep onset REM (SOREM), which has been documented with DORAs, is clearly an undesirable effect, especially for narcoleptic patients and also in fragile populations (e.g. elderly patients) where REM-associated loss of muscle tone may promote an elevated risk of falls. Debate thus remains as to the ideal orexin agent to achieve a balanced increase in REM and non-rapid eye movement (NREM) sleep. Here, we review the evidence that an OX 2 R antagonist should be at least equivalent, or perhaps superior, to a DORA for the treatment of insomnia. An OX 2 R antagonist may produce more balanced sleep than a DORA. Rodent sleep experiments show that the OX 2 R is the primary target of orexin receptor antagonists in sleep modulation. Furthermore, an OX 2 R antagonist should, in theory, have a lower narcoleptic/cataplexic potential. In the clinic, the situation

Activation of the α7 nicotinic ACh receptor induces anxiogenic effects in rats which is blocked by a 5-HT1a receptor antagonist

PubMed Central

Pandya, Anshul A.; Yakel, Jerrel L.

2013-01-01

The α7 nicotinic acetylcholine receptor (nAChR) is highly expressed in different regions of the brain and is associated with cognitive function as well as anxiety. Agonists and positive allosteric modulators (PAMs) of the α7 subtype of nAChRs have been shown to improve cognition. Previously nicotine, which activates both α7 and non-α7 subtypes of nAChRs, has been shown to have an anxiogenic effect in behavioral tests. In this study, we compared the effects of the α7-selective agonist (PNU-282987) and PAM (PNU-120596) in a variety of behavioral tests in Sprague Dawley rats to look at their effects on learning and memory as well as anxiety. We found that neither PNU-282987 nor PNU-120596 improved spatial-learning or episodic memory by themselves. However when cognitive impairment was induced in the rats with scopolamine (1 mg/kg), both PNU-120596 and PNU-282987 were able to reverse this memory impairment and restore it back to normal levels. While PNU-120596 reversed the scopolamine-induced cognitive impairment, it did not have any adverse effect on anxiety. PNU-282987 on the other hand displayed an increase in anxiety-like behavior at a higher dose (10 mg/kg) that was significantly reduced by the serotonin 5-HT1a receptor antagonist WAY-100135. However the α7 receptor antagonist methyllycaconitine was unable to reverse these anxiety-like effects seen with PNU-282987. These results suggest that α7 nAChR PAMs are pharmacologically advantageous over agonists, and should be considered for further development as therapeutic drugs targeting the α7 receptors. PMID:23321689

In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells.

PubMed

Erostegui, C; Norris, C H; Bobbin, R P

1994-04-01

Acetylcholine (ACh) is the major neurotransmitter released from the efferent fibers in the cochlea onto the outer hair cells (OHCs). The type of ACh receptor on OHCs and the events subsequent to receptor activation are unclear. Therefore we studied the effect of agonists and antagonists of the ACh receptor on isolated OHCs from the guinea pig. OHCs were recorded from in whole cell voltage and current clamp configuration. ACh induced an increase in outward K+ current (IACh) which hyperpolarized the OHCs. No desensitization to ACh application was observed. Cs+ replaced K+ in carrying the IACh. The IACh is Ca(2+)-dependent, time and voltage sensitive, and different from the IKCa induced by depolarization of the membrane potential. When tested at 100 microM, several agonists also induced outward current responses (acetylcholine > suberyldicholine > or = carbachol > DMPP) whereas nicotine, cytisine and muscarine did not. The IACh response to 10 microM ACh was blocked by low concentrations of traditional and non-traditional-nicotinic antagonists (strychnine > curare > bicuculline > alpha-bungarotoxin > thimethaphan) and by higher concentrations of muscarinic antagonists (atropine > 4-DAMP > AF-DX 116 > pirenzepine). Pharmacologically, the ACh receptor on OHCs is nicotinic.

The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors.

PubMed

Lochner, Martin; Thompson, Andrew J

2016-09-01

Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [(3)H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

PubMed

Malomouzh, Artem I; Petrov, Konstantin A; Nurullin, Leniz F; Nikolsky, Evgeny E

2015-12-01

Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release. © 2015 International Society for Neurochemistry.

Deletion of muscarinic type 1 acetylcholine receptors alters splenic lymphocyte functions and splenic noradrenaline concentration.

PubMed

Hainke, Susanne; Wildmann, Johannes; Del Rey, Adriana

2015-11-01

The existence of interactions between the immune and the sympathetic nervous systems is well established. Noradrenaline can promote or inhibit the immune response, and conversely, the immune response itself can affect noradrenaline concentration in lymphoid organs, such as the spleen. It is also well known that acetylcholine released by pre-ganglionic neurons can modulate noradrenaline release by the postsynaptic neuron. The spleen does not receive cholinergic innervation, but it has been reported that lymphocytes themselves can produce acetylcholine, and express acetylcholine receptors and acetylcholinesterase. We found that the spleen of not overtly immunized mice in which muscarinic type 1 acetylcholine receptors have been knocked out (M1KO) has higher noradrenaline concentrations than that of the wildtype mice, without comparable alterations in the heart, in parallel to a decreased number of IgG-producing B cells. Splenic lymphocytes from M1KO mice displayed increased in vitro-induced cytotoxicity, and this was observed only when CD4(+) T cells were present. In contrast, heterozygous acetylcholinesterase (AChE+/-) mice, had no alterations in splenic noradrenaline concentration, but the in vitro proliferation of AChE+/- CD4(+) T cells was increased. It is theoretically conceivable that reciprocal effects between neuronally and non-neuronally derived acetylcholine and noradrenaline might contribute to the results reported. Our results emphasize the need to consider the balance between the effects of these mediators for the final immunoregulatory outcome. Copyright © 2015 Elsevier B.V. All rights reserved.

The role of nicotinic acetylcholine and opioid systems of the ventral orbital cortex in modulation of formalin-induced orofacial pain in rats.

PubMed

Yousofizadeh, Shahnaz; Tamaddonfard, Esmaeal; Farshid, Amir Abbas

2015-07-05

Nicotinic acetylcholine and opioid receptors are involved in modulation of pain. In the present study, we investigated the effects of microinjection of nicotinic acetylcholine and opioid compounds into the ventral orbital cortex (VOC) on the formalin-induced orofacial pain in rats. For this purpose, two guide cannulas were placed into the left and right sides of the VOC of the brain. Orofacial pain was induced by subcutaneous injection of a diluted formalin solution (50μl, 1.5%) into the right vibrissa pad and face rubbing durations were recorded at 3-min blocks for 45min. Formalin produced a marked biphasic pain response (first phase: 0-3min and second phase: 15-33min). Epibatidine (a nicotinic receptor agonist) at doses of 0.05, 0.1 and 0.2μg/site, morphine (an opioid receptor agonist) at doses of 0.5, 1 and 2μg/site and their sub-analgesic doses (0.025μg/site epibatidine with 0.25μg/site morphine) combination treatment suppressed the second phase of pain. The antinociceptive effect induced by 0.2μg/site of epibatidine, but not morphine (2μg/site), was prevented by 2μg/site of mecamylamine (a nicotinic receptor antagonist). Naloxone (an opioid receptor antagonist) at a dose of 2μg/site prevented the antinociceptive effects induced by 2μg/site of morphine and 0.2μg/site of epibatidine. No above-mentioned chemical compounds affected locomotor activity. These results showed that at the VOC level, epibatidine and morphine produced antinociception. In addition, opioid receptor might be involved in epibatidine-induced antinociception, but the antinociception induced by morphine was not mediated through nicotinic acetylcholine receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid relief of block by mecamylamine of neuronal nicotinic acetylcholine receptors of rat chromaffin cells in vitro: an electrophysiological and modeling study.

PubMed

Giniatullin, R A; Sokolova, E M; Di Angelantonio, S; Skorinkin, A; Talantova, M V; Nistri, A

2000-10-01

The mechanism responsible for the blocking action of mecamylamine on neuronal nicotinic acetylcholine receptors (nAChRs) was studied on rat isolated chromaffin cells recorded under whole-cell patch clamp. Mecamylamine strongly depressed (IC(50) = 0.34 microM) inward currents elicited by short pulses of nicotine, an effect slowly reversible on wash. The mecamylamine block was voltage-dependent and promptly relieved by a protocol combining membrane depolarization with a nicotine pulse. Either depolarization or nicotine pulses were insufficient per se to elicit block relief. Block relief was transient; response depression returned in a use-dependent manner. Exposure to mecamylamine failed to block nAChRs if they were not activated by nicotine or if they were activated at positive membrane potentials. These data suggest that mecamylamine could not interact with receptors either at rest or at depolarized level. Other nicotinic antagonists like dihydro-beta-erythroidine or tubocurarine did not share this action of mecamylamine although proadifen partly mimicked it. Mecamylamine is suggested to penetrate and block open nAChRs that would subsequently close and trap this antagonist. Computer modeling indicated that the mechanism of mecamylamine blocking action could be described by assuming that 1) mecamylamine-blocked receptors possessed a much slower, voltage-dependent isomerization rate, 2) the rate constant for mecamylamine unbinding was large and poorly voltage dependent. Hence, channel reopening plus depolarization allowed mecamylamine escape and block relief. In the presence of mecamylamine, therefore, nAChRs acquire the new property of operating as coincidence detectors for concomitant changes in membrane potential and receptor occupancy.

Clebopride enhances contractility of the guinea pig stomach by blocking peripheral D2 dopamine receptor and alpha-2 adrenoceptor.

PubMed

Takeda, K; Taniyama, K; Kuno, T; Sano, I; Ishikawa, T; Ohmura, I; Tanaka, C

1991-05-01

The mechanism of action of clebopride on the motility of guinea pig stomach was examined by the receptor binding assay for bovine brain membrane and by measuring gastric contractility and the release of acetylcholine from the stomach. The receptor binding assay revealed that clebopride bound to the D2 dopamine receptor with a high affinity and to the alpha-2 adrenoceptor and 5-HT2 serotonin receptor with relatively lower affinity, and not to D1 dopamine, alpha-1 adrenergic, muscarinic acetylcholine, H1 histamine, or opioid receptor. In strips of the stomach, clebopride at 10(-8) M to 10(-5) M enhanced the electrical transmural stimulation-evoked contraction and the release of acetylcholine. This enhancement was attributed to the blockade of the D2 dopamine receptor and alpha-2 adrenoceptor because: 1) Maximum responses obtained with specific D2 dopamine receptor antagonist, domperidone, and with specific alpha-2 adrenoceptor antagonist, yohimbine, were smaller than that with clebopride, and the sum of the effects of these two specific receptor antagonists is approximately equal to the effect of clebopride. 2) The facilitatory effect of clebopride was partially eliminated by pretreatment of the sample with domperidone or yohimbine, and the facilitatory effect of clebopride was not observed in preparations treated with the combination of domperidone and yohimbine. Clebopride also antagonized the inhibitory effects of dopamine and clonidine on the electrical transmural stimulation-evoked responses. These results indicate that clebopride acts on post ganglionic cholinergic neurons at D2 and alpha-2 receptors in this preparation to enhance enteric nervous system stimulated motility.

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

PubMed

Maggi, C A; Patacchini, R; Giuliani, S; Rovero, P; Dion, S; Regoli, D; Giachetti, A; Meli, A

1990-07-01

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA. Similar pA2 values were obtained after 15 or 90min of incubation with the antagonists. Peptides I, II and III had no inhibitory effect on contractions produced by noradrenaline in the RPA or by carbachol in the HT. 5. Peptides I, II and III showed weak or no antagonistic activity toward the vasodilatator effect of substance P in the dog carotid artery (NK, receptor

Cholinesterases: structure of the active site and mechanism of the effect of cholinergic receptor blockers on the rate of interaction with ligands

NASA Astrophysics Data System (ADS)

Antokhin, A. M.; Gainullina, E. T.; Taranchenko, V. F.; Ryzhikov, S. B.; Yavaeva, D. K.

2010-10-01

Modern views on the structure of cholinesterase active sites and the mechanism of their interaction with organophosphorus inhibitors are considered. The attention is focused on the mechanism of the effect of cholinergic receptor blockers, acetylcholine antagonists, on the rate of interaction of acetylcholine esterase with organophosphorus inhibitors.

Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

PubMed

Santafe, M M; Priego, M; Obis, T; Garcia, N; Tomàs, M; Lanuza, M A; Tomàs, J

2015-07-01

Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Probing for and Quantifying Agonist Hydrogen Bonds in α6β2 Nicotinic Acetylcholine Receptors.

PubMed

Post, Michael R; Lester, Henry A; Dougherty, Dennis A

2017-04-04

Designing subtype-selective agonists for neuronal nicotinic acetylcholine receptors is a challenging and significant goal aided by intricate knowledge of each subtype's binding patterns. We previously reported that in α6β2 receptors, acetylcholine makes a functional cation-π interaction with Trp149, but nicotine and TC299423 do not, suggesting a distinctive binding site. This work explores hydrogen binding at the backbone carbonyl associated with α6β2 Trp149. Substituting residue i + 1, Thr150, with its α-hydroxy analogue (Tah) attenuates the carbonyl's hydrogen bond accepting ability. At α6(T150Tah)β2, nicotine shows a 24-fold loss of function, TC299423 shows a modest loss, and acetylcholine shows no effect. Nicotine was further analyzed via a double-mutant cycle analysis utilizing N'-methylnicotinium, which indicated a hydrogen bond in α6β2 with a ΔΔG of 2.6 kcal/mol. Thus, even though nicotine does not make the conserved cation-π interaction with Trp149, it still makes a functional hydrogen bond to its associated backbone carbonyl.

σ Receptor antagonist attenuation of methamphetamine-induced neurotoxicity is correlated to body temperature modulation.

PubMed

Robson, Matthew J; Seminerio, Michael J; McCurdy, Christopher R; Coop, Andrew; Matsumoto, Rae R

2013-01-01

Methamphetamine (METH) causes hyperthermia and dopaminergic neurotoxicity in the rodent striatum. METH interacts with σ receptors and σ receptor antagonists normally mitigate METH-induced hyperthermia and dopaminergic neurotoxicity. The present study was undertaken because in two experiments, pretreatment with σ receptor antagonists failed to attenuate METH-induced hyperthermia in mice. This allowed us to determine whether the ability of σ receptor antagonists (AZ66 and AC927) to mitigate METH-induced neurotoxicity depends upon their ability to modulate METH-induced hyperthermia. Mice were treated using a repeated dosing paradigm and body temperatures recorded. Striatal dopamine was measured one week post-treatment. The data indicate that the ability of σ receptor antagonists to attenuate METH-induced dopaminergic neurotoxicity is linked to their ability to block METH-induced hyperthermia. The ability of σ receptor antagonists to mitigate METH-induced hyperthermia may contribute to its neuroprotective actions.

Acetylcholine is released from taste cells, enhancing taste signalling

PubMed Central

Dando, Robin; Roper, Stephen D

2012-01-01

Acetylcholine (ACh), a candidate neurotransmitter that has been implicated in taste buds, elicits calcium mobilization in Receptor (Type II) taste cells. Using RT-PCR analysis and pharmacological interventions, we demonstrate that the muscarinic acetylcholine receptor M3 mediates these actions. Applying ACh enhanced both taste-evoked Ca2+ responses and taste-evoked afferent neurotransmitter (ATP) secretion from taste Receptor cells. Blocking muscarinic receptors depressed taste-evoked responses in Receptor cells, suggesting that ACh is normally released from taste cells during taste stimulation. ACh biosensors confirmed that, indeed, taste Receptor cells secrete acetylcholine during gustatory stimulation. Genetic deletion of muscarinic receptors resulted in significantly diminished ATP secretion from taste buds. The data demonstrate a new role for acetylcholine as a taste bud transmitter. Our results imply specifically that ACh is an autocrine transmitter secreted by taste Receptor cells during gustatory stimulation, enhancing taste-evoked responses and afferent transmitter secretion. PMID:22570381

Selective activation of α7 nicotinic acetylcholine receptor by PHA-543613 improves Aβ25-35-mediated cognitive deficits in mice.

PubMed

Sadigh-Eteghad, S; Talebi, M; Mahmoudi, J; Babri, S; Shanehbandi, D

2015-07-09

Agonists of α7 nicotinic acetylcholine receptors (nAChRs) are currently being considered as therapeutic approaches for managing cognitive deficits in Alzheimer's disease (AD). Present study was designed to evaluate the effect of α7 nAChR selective activation by PHA-543613 (PHA) on beta-amyloid (Aβ)25-35-mediated cognitive deficits in mice. For this purpose, PHA (1mg/kg, i.p.), a selective α7 nAChR agonist, and galantamine (Gal) (3mg/kg, s.c.), an acetylcholine-esterase inhibitor (AChEI) effects on α7 nAChR were tested in Aβ25-35-received (intracerebroventricular, 10 nmol) mice model of AD. Methyllycaconitine (MLA) (1mg/kg, i.p.), a α7 nAChR antagonist, was used for receptor blockage effects evaluation. Working and reference memory in animals was assessed by the Morris water maze (MWM) task. The mRNA and protein levels of α7 subunit were analyzed by real-time PCR and Western blotting, respectively. PHA and Gal, ameliorate Aβ-impaired working and reference memory. However, Gal had less effect than PHA in this regard. Pretreatment with MLA reverses both Gal and PHA effects in MWM. PHA and Gal treatment prevent Aβ-induced α7 subunit protein reduction, but Gal has lesser effect than PHA. This effect blocked by pretreatment with MLA. In neither the pretreatment nor treatment group, the mRNA levels of nAChR α7 subunit were significantly changed. Therefore, α7 nAChR activation, reduces Aβ-induced cognitive deficits and increases the α7 protein level and subsequent neuron survival. However, blockage of receptor, increases Aβ toxicity and cognitive impairment and reduces the α7 nAChR protein level and flowing neuroprotection. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Impulsive behavior and nicotinic acetylcholine receptors.

PubMed

Ohmura, Yu; Tsutsui-Kimura, Iku; Yoshioka, Mitsuhiro

2012-01-01

Higher impulsivity is thought to be a risk factor for drug addiction, criminal involvement, and suicide. Excessive levels of impulsivity are often observed in several psychiatric disorders including attention-deficit/hyperactivity disorder and schizophrenia. Previous studies have demonstrated that nicotinic acetylcholine receptors (nAChRs) are involved in impulsive behavior. Here, we introduce recent advances in this field and describe the role of the following nAChR-related brain mechanisms in modulating impulsive behavior: dopamine release in the ventral striatum; α4β2 nAChRs in the infralimbic cortex, which is a ventral part of the medial prefrontal cortex (mPFC); and dopamine release in the mPFC. We also suggest several potential therapeutic drugs to address these mechanisms in impulsivity-related disorders and explore future directions to further elucidate the roles of central nAChRs in impulsive behavior.

Melanocortin Antagonist Tetrapeptides with Minimal Agonist Activity at the Mouse Melanocortin-3 Receptor

PubMed Central

2014-01-01

The melanocortin system regulates many important functions in the body. There are five melanocortin G protein-coupled receptor subtypes known to date. Herein, we report a structure–activity relationship (SAR) study of a tetrapeptide lead discovered through a double substitution strategy at the melanocortin core His-Phe-Arg-Trp sequence. Several compounds were identified with micromolar agonist activity at the mouse melanocortin-1 (mMC1R) and mouse melanocortin-5 receptor (mMC5R) subtypes, weak antagonist activity at the mouse melanocortin-3 receptor (mMC3R), and potent antagonist activity at the mouse melanocortin-4 receptor (mMC4R). Two compounds (2 and 3) were nanomolar mMC4R antagonists with no mMC3R antagonist activity observed. Additionally, we identified three tetrapeptide MC3R antagonists (1, 6, and 7) that possess minimal mMC3R agonist activity only at 100 μM, not commonly observed for mMC3R/mMC4R antagonists. These novel molecular templates have the potential as molecular probes to better differentiate the roles of the centrally expressed MC3 and MC4 receptors. PMID:25699138

Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Newsom-Davis, J.; Willcox, N.; Calder, L.

1981-11-26

We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlatedmore » with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.« less

Stimulation of D2 receptors in the prefrontal cortex reduces PCP-induced hyperactivity, acetylcholine release and dopamine metabolism in the nucleus accumbens.

PubMed

Del Arco, A; Mora, F; Mohammed, A H; Fuxe, K

2007-02-01

The aim of the present study was to investigate the effects of stimulation of D2 receptors in the prefrontal cortex (PFC) on spontaneous motor activity and the hyperactivity induced by the psychomimetic phencyclidine (PCP). In addition, the effects of prefrontal D2 stimulation under PCP treatment on dialysate concentrations of acetylcholine, choline, dopamine, DOPAC and HVA in the nucleus accumbens were also investigated. Sprague-Dawley male rats were implanted with guide cannulae to perform bilateral injections into the medial PFC of the D2 agonist quinpirole (1.5 and 5 microg/side). Horizontal and vertical spontaneous motor activity and the motor activity induced by systemic injections of the PCP (5 mg/kg i.p.) were monitored in the open field. PFC injections of quinpirole (1.5 and 5 microg/side) significantly decreased horizontal and vertical spontaneous motor activity in a dose-related manner. These effects were blocked by the D2 antagonist raclopride (5 microg/side). Microinjections of quinpirole (1.5 and 5 microg/side) into the PFC also significantly attenuated the hyperactivity produced by PCP (5 mg/kg i.p.). PCP also increased dialysate concentrations of acetylcholine, and dopamine metabolites in the nucleus accumbens. These increases were also reduced by injections of quinpirole (5 microg/side) into the PFC. These results suggest that the stimulation of prefrontal D2 receptors plays an inhibitory role in regulating spontaneous and PCP-induced motor activity and also in the neurochemical changes produced by PCP in the nucleus accumbens.

Monkey Adrenal Chromaffin Cells Express α6β4* Nicotinic Acetylcholine Receptors

PubMed Central

Scadden, Mick´l; Carmona-Hidalgo, Beatriz; McIntosh, J. Michael; Albillos, Almudena

2014-01-01

Nicotinic acetylcholine receptors (nAChRs) that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs. PMID:24727685

Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and {beta}-adrenergic receptor signaling pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shin, Vivian Yvonne; Jin, H.C.; Ng, Enders K.O.

Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor ({alpha}7 nAChR) and {beta}-adrenergic receptors. Treatment of cells with {alpha}-bungarotoxin ({alpha}-BTX, {alpha}7nAChR antagonist) or propranolol ({beta}-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE{sub 2} and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE{sub 2} induction canmore » only be suppressed by propranolol, but not {alpha}-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.« less

5-HT7 Receptor Antagonists with an Unprecedented Selectivity Profile.

PubMed

Ates, Ali; Burssens, Pierre; Lorthioir, Olivier; Lo Brutto, Patrick; Dehon, Gwenael; Keyaerts, Jean; Coloretti, Francis; Lallemand, Bénédicte; Verbois, Valérie; Gillard, Michel; Vermeiren, Céline

2018-04-23

Selective leads: In this study, we generated a new series of serotonin 5-HT 7 receptor antagonists. Their synthesis, structure-activity relationships, and selectivity profiles are reported. This series includes 5-HT 7 antagonists with unprecedented high selectivity for the 5-HT 7 receptor, setting the stage for lead optimization of drugs acting on a range of neurological targets. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.

PubMed

Gautam, Dinesh; Han, Sung-Jun; Hamdan, Fadi F; Jeon, Jongrye; Li, Bo; Li, Jian Hua; Cui, Yinghong; Mears, David; Lu, Huiyan; Deng, Chuxia; Heard, Thomas; Wess, Jürgen

2006-06-01

One of the hallmarks of type 2 diabetes is that pancreatic beta cells fail to release sufficient amounts of insulin in the presence of elevated blood glucose levels. Insulin secretion is modulated by many hormones and neurotransmitters including acetylcholine, the major neurotransmitter of the peripheral parasympathetic nervous system. The physiological role of muscarinic acetylcholine receptors expressed by pancreatic beta cells remains unclear at present. Here, we demonstrate that mutant mice selectively lacking the M3 muscarinic acetylcholine receptor subtype in pancreatic beta cells display impaired glucose tolerance and greatly reduced insulin release. In contrast, transgenic mice selectively overexpressing M3 receptors in pancreatic beta cells show a profound increase in glucose tolerance and insulin release. Moreover, these mutant mice are resistant to diet-induced glucose intolerance and hyperglycemia. These findings indicate that beta cell M3 muscarinic receptors play a key role in maintaining proper insulin release and glucose homeostasis.

Crystal structure of human glycine receptor-α3 bound to antagonist strychnine.

PubMed

Huang, Xin; Chen, Hao; Michelsen, Klaus; Schneider, Stephen; Shaffer, Paul L

2015-10-08

Neurotransmitter-gated ion channels of the Cys-loop receptor family are essential mediators of fast neurotransmission throughout the nervous system and are implicated in many neurological disorders. Available X-ray structures of prokaryotic and eukaryotic Cys-loop receptors provide tremendous insights into the binding of agonists, the subsequent opening of the ion channel, and the mechanism of channel activation. Yet the mechanism of inactivation by antagonists remains unknown. Here we present a 3.0 Å X-ray structure of the human glycine receptor-α3 homopentamer in complex with a high affinity, high-specificity antagonist, strychnine. Our structure allows us to explore in detail the molecular recognition of antagonists. Comparisons with previous structures reveal a mechanism for antagonist-induced inactivation of Cys-loop receptors, involving an expansion of the orthosteric binding site in the extracellular domain that is coupled to closure of the ion pore in the transmembrane domain.

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

PubMed

Mochizuki, Michiyo; Kori, Masakuni; Kobayashi, Katsumi; Yano, Takahiko; Sako, Yuu; Tanaka, Maiko; Kanzaki, Naoyuki; Gyorkos, Albert C; Corrette, Christopher P; Cho, Suk Young; Pratt, Scott A; Aso, Kazuyoshi

2016-03-24

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grandič, Marjana; Aráoz, Romulo; Molgó, Jordi

APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC{sub 50} = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials,more » recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC{sub 50} = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1{sub 2}β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC{sub 50} = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1{sub 2}β1γδ) than for the mouse (α1{sub 2}β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.« less

Positive allosteric modulators as an approach to nicotinic acetylcholine receptor- targeted therapeutics: advantages and limitations

PubMed Central

Williams, Dustin K.; Wang, Jingyi; Papke, Roger L.

2011-01-01

Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues. PMID:21575610

Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.

PubMed

Williams, Dustin K; Wang, Jingyi; Papke, Roger L

2011-10-15

Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues. Copyright © 2011 Elsevier Inc. All rights reserved.

Schizophrenia and the alpha7 nicotinic acetylcholine receptor.

PubMed

Martin, Laura F; Freedman, Robert

2007-01-01

In addition to the devastating symptoms of psychosis, many people with schizophrenia also suffer from cognitive impairment. These cognitive symptoms lead to marked dysfunction and can impact employability, treatment adherence, and social skills. Deficits in P50 auditory gating are associated with attentional impairment and may contribute to cognitive symptoms and perceptual disturbances. This nicotinic cholinergic-mediated inhibitory process represents a potential new target for therapeutic intervention in schizophrenia. This chapter will review evidence implicating the nicotinic cholinergic, and specifically, the alpha7 nicotinic receptor system in the pathology of schizophrenia. Impaired auditory sensory gating has been linked to the alpha7 nicotinic receptor gene on the chromosome 15q14 locus. A majority of persons with schizophrenia are heavy smokers. Although nicotine can acutely reverse diminished auditory sensory gating in people with schizophrenia, this effect is lost on a chronic basis due to receptor desensitization. The alpha7 nicotinic agonist 3-(2,4 dimethoxy)benzylidene-anabaseine (DMXBA) can also enhance auditory sensory gating in animal models. DMXBA is well tolerated in humans and a new study in persons with schizophrenia has found that DMXBA enhances both P50 auditory gating and cognition. alpha7 Nicotinic acetylcholine receptor agonists appear to be viable candidates for the treatment of cognitive disturbances in schizophrenia.

Nicotine-Induced Effects on Nicotinic Acetylcholine Receptors (nAChRs), Ca2+ and Brain-Derived Neurotrophic Factor (BDNF) in STC-1 Cells.

PubMed

Qian, Jie; Mummalaneni, Shobha K; Alkahtani, Reem M; Mahavadi, Sunila; Murthy, Karnam S; Grider, John R; Lyall, Vijay

2016-01-01

In addition to the T2R bitter taste receptors, neuronal nicotinic acetylcholine receptors (nAChRs) have recently been shown to be involved in the bitter taste transduction of nicotine, acetylcholine and ethanol. However, at present it is not clear if nAChRs are expressed in enteroendocrine cells other than beta cells of the pancreas and enterochromaffin cells, and if they play a role in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of nAChRs in enteroendocrine STC-1 cells. Our studies using RT-PCR, qRT-PCR, immunohistochemical and Western blotting techniques demonstrate that STC-1 cells express several α and β nAChR subunits. Exposing STC-1 cells to nicotine acutely (24h) or chronically (4 days) induced a differential increase in the expression of nAChR subunit mRNA and protein in a dose- and time-dependent fashion. Mecamylamine, a non-selective antagonist of nAChRs, inhibited the nicotine-induced increase in mRNA expression of nAChRs. Exposing STC-1 cells to nicotine increased intracellular Ca2+ in a dose-dependent manner that was inhibited in the presence of mecamylamine or dihydro-β-erythroidine, a α4β2 nAChR antagonist. Brain-derived neurotrophic factor (BDNF) mRNA and protein were detected in STC-1 cells using RT-PCR, specific BDNF antibody, and enzyme-linked immunosorbent assay. Acute nicotine exposure (30 min) decreased the cellular content of BDNF in STC-1 cells. The nicotine-induced decrease in BDNF was inhibited in the presence of mecamylamine. We also detected α3 and β4 mRNA in intestinal mucosal cells and α3 protein expression in intestinal enteroendocrine cells. We conclude that STC-1 cells and intestinal enteroendocrine cells express nAChRs. In STC-1 cells nAChR expression is modulated by exposure to nicotine in a dose- and time-dependent manner. Nicotine interacts with nAChRs and inhibits BDNF expression in STC-1 cells.

Rapid synthesis of acetylcholine receptors at neuromuscular junctions.

PubMed

Ramsay, D A; Drachman, D B; Pestronk, A

1988-10-11

The rate of acetylcholine receptor (AChR) degradation in mature, innervated mammalian neuromuscular junctions has recently been shown to be biphasic; up to 20% are rapidly turned over (RTOs; half life less than 1 day) whereas the remainder are lost more slowly ('stable' AChRs; half life 10-12 days). In order to maintain normal junctional receptor density, synthesis and insertion of AChRs should presumably be sufficiently rapid to replace both the RTOs and the stable receptors. We have tested this prediction by blocking pre-existing AChRs in the mouse sternomastoid muscle with alpha-bungarotoxin (alpha-BuTx), and monitoring the subsequent appearance of 'new' junctional AChRs at intervals of 3 h to 20 days by labeling them with 125I-alpha-BuTx. The results show that new receptors were initially inserted rapidly (16% at 24 h and 28% at 48 h). The rate of increase of 'new' 125I-alpha-BuTx binding sites gradually slowed down during the remainder of the time period studied. Control observations excluded possible artifacts of the experimental procedure including incomplete blockade of AChRs, dissociation of toxin-receptor complexes, or experimentally induced alteration of receptor synthesis. The present demonstration of rapid synthesis and incorporation of AChRs at innervated neuromuscular junctions provides support for the concept of a subpopulation of rapidly turned over AChRs. The RTOs may serve as precursors for the larger population of stable receptors and have an important role in the metabolism of the neuromuscular synapse.

Unorthodox Acetylcholine Binding Sites Formed by α5 and β3 Accessory Subunits in α4β2* Nicotinic Acetylcholine Receptors.

PubMed

Jain, Akansha; Kuryatov, Alexander; Wang, Jingyi; Kamenecka, Theodore M; Lindstrom, Jon

2016-11-04

All nicotinic acetylcholine receptors (nAChRs) evolved from homomeric nAChRs in which all five subunits are involved in forming acetylcholine (ACh) binding sites at their interfaces. Heteromeric α4β2* nAChRs typically have two ACh binding sites at α4/β2 interfaces and a fifth accessory subunit surrounding the central cation channel. β2 accessory subunits do not form ACh binding sites, but α4 accessory subunits do at the α4/α4 interface in (α4β2) 2 α4 nAChRs. α5 and β3 are closely related subunits that had been thought to act only as accessory subunits and not take part in forming ACh binding sites. The effect of agonists at various subunit interfaces was determined by blocking homologous sites at these interfaces using the thioreactive agent 2-((trimethylammonium)ethyl) methanethiosulfonate (MTSET). We found that α5/α4 and β3/α4 interfaces formed ACh binding sites in (α4β2) 2 α5 and (α4β2) 2 β3 nAChRs. The α4/α5 interface in (β2α4) 2 α5 nAChRs also formed an ACh binding site. Blocking of these sites with MTSET reduced the maximal ACh evoked responses of these nAChRs by 30-50%. However, site-selective agonists NS9283 (for the α4/α4 site) and sazetidine-A (for the α4/β2 site) did not act on the ACh sites formed by the α5/α4 or β3/α4 interfaces. This suggests that unorthodox sites formed by α5 and β3 subunits have unique ligand selectivity. Agonists or antagonists for these unorthodox sites might be selective and effective drugs for modulating nAChR function to treat nicotine addiction and other disorders. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

From Chemotherapy-Induced Emesis to Neuroprotection: Therapeutic Opportunities for 5-HT3 Receptor Antagonists.

PubMed

Fakhfouri, Gohar; Mousavizadeh, Kazem; Mehr, Sharam Ejtemaei; Dehpour, Ahmad Reza; Zirak, Mohammad Reza; Ghia, Jean-Eric; Rahimian, Reza

2015-12-01

5-HT3 receptor antagonists are extensively used as efficacious agents in counteracting chemotherapy-induced emesis. Recent investigations have shed light on other potential effects (analgesic, anxiolytic, and anti-psychotic). Some studies have reported neuroprotective properties for the 5-HT3 receptor antagonists in vitro and in vivo. When administered to Aβ-challenged rat cortical neurons, 5-HT3 receptor antagonists substantially abated apoptosis, elevation of cytosolic Ca(2), glutamate release, reactive oxygen species (ROS) generation, and caspase-3 activity. In addition, in vivo studies show that 5-HT3 receptor antagonists possess, alongside their anti-emetic effects, notable immunomodulatory properties in CNS. We found that pretreatment with tropisetron significantly improved neurological deficits and diminished leukocyte transmigration into the brain, TNF-α level, and brain infarction in a murine model of embolic stroke. Our recent investigation revealed that tropisetron protects against Aβ-induced neurotoxicity in vivo through both 5-HT3 receptor-dependent and -independent pathways. Tropisetron, in vitro, was found to be an efficacious inhibitor of the signaling pathway leading to the activation of pro-inflammatory NF-κB, a transcription factor pivotal to the upregulation of several neuroinflammatory mediators in brain. This mini review summarizes novel evidence concerning effects of 5-HT3 antagonists and their possible mechanisms of action in ameliorating neurodegenerative diseases including Alzheimer, multiple sclerosis, and stroke. Further, we discuss some newly synthesized 5-HT3 receptor antagonists with dual properties of 5-HT3 receptor blockade/alpha-7 nicotinic receptor activator and their potential in management of memory impairment. Since 5-HT3 receptor antagonists possess a large therapeutic window, they can constitute a scaffold for design and synthesis of new neuroprotective medications.

Discovery of spiropiperidine-based potent and selective Orexin-2 receptor antagonists.

PubMed

Fujimoto, Tatsuhiko; Tomata, Yoshihide; Kunitomo, Jun; Hirozane, Mariko; Marui, Shogo

2011-11-01

To generate novel human Orexin-2 Receptor (OX2R) antagonists, a spiropiperidine based scaffold was designed and a SAR study was carried out. Compound 4f possessed the highest OX2R antagonistic activity with an IC(50) value of 3nM with 450-fold selectivity against Orexin-1 Receptor (OX1R). Copyright © 2011 Elsevier Ltd. All rights reserved.

Role of muscarinic receptor antagonists in urgency and nocturia.

PubMed

Michel, Martin C; de la Rosette, Jean J M C H

2005-09-01

The overactive bladder (OAB) syndrome is defined as urgency, with or without urgency incontinence, usually accompanied by frequency and nocturia. Muscarinic receptor antagonists are the most established form of treatment for OAB, but until recently their effectiveness was only confirmed for symptoms of incontinence and frequency. In recent studies, selected muscarinic antagonists, including darifenacin, solifenacin, tolterodine and trospium, significantly reduced the number of urgency episodes per day relative to placebo. While some data raise the possibility that certain of these agents may be more effective than others in this regard, this variability in their effect on urgency needs to be confirmed in future studies. Moreover, it remains to be determined whether counting the number of urgency episodes or assessing the subjective intensity of the sensation of urgency more adequately reflects patient needs and therapeutic efficacy. For nocturia, muscarinic receptor antagonists have only inconsistently shown statistically greater effects than placebo. This inconsistency may relate to the multifactorial nature of nocturia, which even in patients with OAB can have many causes, not all of which may respond/be sensitive to muscarinic receptor antagonism.

Interaction between Antagonist of Cannabinoid Receptor and Antagonist of Adrenergic Receptor on Anxiety in Male Rat.

PubMed

Komaki, Alireza; Abdollahzadeh, Fatemeh; Sarihi, Abdolrahman; Shahidi, Siamak; Salehi, Iraj

2014-01-01

Anxiety is among the most common and treatable mental disorders. Adrenergic and cannabinoid systems have an important role in the neurobiology of anxiety. The elevated plus-maze (EPM) has broadly been used to investigate anxiolytic and anxiogenic compounds. The present study investigated the effects of intraperitoneal (IP) injection of cannabinoid CB1 receptor antagonist (AM251) in the presence of alpha-1 adrenergic antagonist (Prazosin) on rat behavior in the EPM. In this study, the data were obtained from male Wistar rat, which weighing 200- 250 g. Animal behavior in EPM were videotaped and saved in computer for 10 min after IP injection of saline, AM251 (0.3 mg/kg), Prazosin (0.3 mg/kg) and AM251 + Prazosin, subsequently scored for conventional indices of anxiety. During the test period, the number of open and closed arms entries, the percentage of entries into the open arms of the EPM, and the spent time in open and closed arms were recorded. Diazepam was considered as a positive control drug with anxiolytic effect (0.3, 0.6, 1.2 mg/kg). Diazepam increased the number of open arm entries and the percentage of spent time on the open arms. IP injection of AM251 before EPM trial decreased open arms exploration and open arm entry. Whereas, Prazosin increased open arms exploration and open arm entry. This study showed that both substances in simultaneous injection have conflicting effects on the responses of each of these two compounds in a single injection. Injection of CB1 receptor antagonist may have an anxiogenic profile in rat, whereas adrenergic antagonist has an anxiolytic effect. Further investigations are essential for better understanding of anxiolytic and anxiogenic properties and neurobiological mechanisms of action and probable interactions of the two systems.

Microinjection of acetylcholine into cerebellar fastigial nucleus induces blood depressor response in anesthetized rats.

PubMed

Zhang, Changzheng; Luo, Wen; Zhou, Peiling; Sun, Tingzhe

2016-08-26

It is well known that the cerebellar fastigial nucleus (FN) is involved in cardiovascular modulation, and has direct evidence of cholinergic activity; however, whether and how acetylcholine (ACh) in the FN modulates blood pressure has not been investigated. In this study, we analyzed mean arterial pressure, maximal change in mean arterial pressure, and the reaction time of blood pressure changes after microinjection of cholinergic reagents into the FN in anesthetized rats. The results showed that ACh evoked a concentration-dependent (10, 30 and 100mM) effect on blood pressure down-regulation. The muscarinic ACh (mACh) receptor antagonist atropine, but not the nicotinic ACh (nACh) receptor antagonist mecamylamine, blocked the ACh-mediated depressor response. The mACh receptor agonist oxotremorine M, rather than nACh receptor agonist nicotine, mimicked the ACh-mediated blood pressure decrease in a dose-dependent manner (10, 30 and 100mM). These results indicate that cholinergic input in the cerebellar FN exerts a depressor effect on systemic blood pressure regulation, and such effects are substantially contributed by mACh rather than nACh receptors, although the precise mechanism concerning the role of mACh receptor in FN-mediated blood pressure modulation remains to be elucidated. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Functional evidence for the inflammatory reflex in teleosts: A novel α7 nicotinic acetylcholine receptor modulates the macrophage response to dsRNA.

PubMed

Torrealba, Débora; Balasch, Joan Carles; Criado, Manuel; Tort, Lluís; Mackenzie, Simon; Roher, Nerea

2018-07-01

The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (α7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost α7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout α7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind α-bungarotoxin (α-BTX), a competitive antagonist for α7nAChRs. Moreover, nicotine stimulation produces a decrease in pro-inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional α7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enhancement effects of nicotine on neurogenic relaxation responses in the corpus cavernosum in rabbits: the role of nicotinic acetylcholine receptor subtypes.

PubMed

Ozturk Fincan, Gokce Sevim; Vural, Ismail Mert; Ercan, Zeynep Sevim; Sarioglu, Yusuf

2010-02-10

Nicotine acts as an agonist of nicotinic acetylcholine receptors, which belong to a superfamily of neurotransmitter-gated ion channels. We previously demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked nitrergic relaxation responses via activation of nicotinic acetylcholine receptors. The aim of the present study is to investigate the subtypes of nicotinic acetylcholine receptors in rabbit corpus cavernosum. EFS-evoked relaxation responses were recorded from corpus cavernosum strips obtained from rabbits with an isometric force displacement transducers. Effects of nicotine on EFS-evoked relaxations were examined in pre-contracted tissues. Then the effect of nicotine on the EFS-evoked relaxations was examined in the presence of hexamethonium, dihydro-beta-erythroidine, mecamylamine or alpha-bungarotoxin. In our study, nicotine (3 x 10(-5), 10(-4)) transiently increased nitrergic relaxations induced by EFS in the rabbit isolated corpus cavernosum. While hexamethonium and mecamylamine near totally inhibited or abolished the neurorelaxation response to nicotine (3 x 10(-5)) on EFS, dihydro-beta-erythroidine and alpha-bungarotoxin partially inhibited these responses. These findings demonstrated that the alpha3-beta4, alpha4-beta2 and alpha7 subunits of nicotinic acetylcholine receptors play role on the nicotine-induced augmentation in EFS-evoked relaxation responses in rabbit corpus cavernosum. Copyright (c) 2009 Elsevier B.V. All rights reserved.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

PubMed

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-04-01

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges.

Functional antagonistic properties of clozapine at the 5-HT3 receptor.

PubMed

Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

1996-08-23

The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

The 5-HT2A receptor antagonist M100907 is more effective in counteracting NMDA antagonist- than dopamine agonist-induced hyperactivity in mice.

PubMed

Carlsson, M L; Martin, P; Nilsson, M; Sorensen, S M; Carlsson, A; Waters, S; Waters, N

1999-01-01

The purpose of the present study was to compare the effectiveness of the selective 5-HT2A antagonist M100907 in different psychosis models. The classical neuroleptic haloperidol was used as reference compound. Two hyperdopaminergia and two hypoglutamatergia mouse models were used. Hyperdopaminergia was produced by the DA releaser d-amphetamine or the DA uptake inhibitor GBR 12909. Hypoglutamatergia was produced by the un-competitive NMDA receptor antagonist MK-801 or the competitive NMDA receptor antagonist D-CPPene. M100907 was found to counteract the locomotor stimulant effects of the NMDA receptor antagonists MK-801 and D-CPPene, but spontaneous locomotion, d-amphetamine- and GBR-12909-induced hyperactivity were not significantly affected. Haloperidol, on the other hand, antagonized both NMDA antagonist- and DA agonist-induced hyperactivity, as well as spontaneous locomotion in the highest dose used. Based on the present and previous results we draw the conclusion that 5-HT2A receptor antagonists are particularly effective against behavioural anomalies resulting from hypoglutamatergia of various origins. The clinical implications of our results and conclusions would be that a 5-HT2A receptor antagonist, due to i a the low side effect liability, could be the preferable treatment strategy in various disorders associated with hypoglutamatergia; such conditions might include schizophrenia, childhood autism and dementia disorders.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist.

PubMed

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W; Vanden Broeck, Jozef; Tourwé, Dirk

2011-04-14

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3',5'-(CF(3))(2)-Bn], 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], and 23 [Ac-Tic-NMe-3',5'-(CF(3))(2)-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3',5'-(CF(3))(2)-Bn], which combines the N terminus of the established Dmt(1)-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH(2)) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH(2) (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist

PubMed Central

Ballet, Steven; Feytens, Debby; Buysse, Koen; Chung, Nga N.; Lemieux, Carole; Tumati, Suneeta; Keresztes, Attila; Van Duppen, Joost; Lai, Josephine; Varga, Eva; Porreca, Frank; Schiller, Peter W.; Broeck, Jozef Vanden; Tourwé, Dirk

2011-01-01

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′,5′-(CF3)2-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn] and 23 [Ac-Tic-NMe-3′,5′-(CF3)2-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-D-Arg-Aba-Gly-NMe-3′,5′-(CF3)2-Bn], which combines the N-terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, i.e. Dmt-D-Arg-Aba-Gly-NH2 36, also proved to be an extremely potent and balanced μ- and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity. PMID:21413804

Vector-averaged gravity does not alter acetylcholine receptor single channel properties

NASA Technical Reports Server (NTRS)

Reitstetter, R.; Gruener, R.

1994-01-01

To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

Activation and modulation of human α4β2 nicotinic acetylcholine receptors by the neonicotinoids clothianidin and imidacloprid

PubMed Central

Li, Ping; Ann, Jason; Akk, Gustav

2013-01-01

Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. While highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1–4 % of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI to the human α4β2 receptor is 18 µM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)2(β2)3 receptors significantly more strongly inhibited than the (α4)3(β2)2 receptors. PMID:21538459

Rational Design of Potent Antagonists to the Human Growth Hormone Receptor

NASA Astrophysics Data System (ADS)

Fuh, Germaine; Cunningham, Brian C.; Fukunaga, Rikiro; Nagata, Shigekazu; Goeddel, David V.; Wells, James A.

1992-06-01

A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially-a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

Muscarinic acetylcholine M4 receptors play a critical role in oxotremorine-induced DARPP-32 phosphorylation at threonine 75 in isolated medium spiny neurons.

PubMed

Liu, Liqun; Huang, Yuqi; Huang, Qing; Zhao, Zhe; Yu, Jianqiang; Wang, Liyun

2017-05-01

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) play essential roles in dopamine (DA) transmission in the striatum. It is suggested that a link exists between muscarinic acetylcholine receptors (mAChRs) and DA/DARPP-32 signaling, but the molecular mechanisms mediating this relationship have not been elucidated. The predominant mAChRs subtypes in the striatum are M 1 and M 4 . In this study, we investigated the functions of these two receptors, particularly M 4 , in regulating cAMP production and DARPP-32 phosphorylation in rat striatal medium spiny neurons (MSNs). We used time-resolved fluorescence resonance energy transfer, immunofluorescence confocal microscopy, and western blot assays. In cultured intact MSNs, we confirmed that muscarinic M 1 and M 4 receptors were highly expressed. Notably, M 4 receptors were co-expressed with D 1 receptors in only a portion of the cultured MSNs. The nonselective muscarinic agonist oxotremorine M (OX) slightly enhanced cAMP production, but this effect was independent of M 1 or M 4 receptors. However, OX directly participated in DARPP-32 phosphorylation, phosphorylating DARPP-32 at Thr75 (the CDK5 site) and concomitantly de-phosphorylating DARPP-32 at Thr34 (the PKA site) in virtually cultured MSNs, whereas APO phosphorylated DARPP-32 at both Thr34 and Thr75. The OX-induced time-dependent increase in DARPP-32 phosphorylation at Thr75 was accompanied by increased p35 and CDK5 activity. Specifically, elevated immunoreactivity for phospho-DARPP-32-Thr75 and p35 was detected in M 4 receptor-expressing MSNs. Both genetic knockdown and pharmacologic inhibition of M 4 receptors with MT3, an M 4 receptor-selective antagonist, decreased the OX-induced DARPP-32-Thr75 phosphorylation in MSNs. These results indicate that the M 4 muscarinic receptor plays a critical role in modulating phosphorylation of DARPP-32-Thr75 in MSNs. The results suggest that M 4 receptor activation acts antagonistically with dopamine D 1 -like

Anti-idiotypic antibody: A new strategy for the development of a growth hormone receptor antagonist.

PubMed

Lan, Hainan; Zheng, Xin; Khan, Muhammad Akram; Li, Steven

2015-11-01

In general, traditional growth hormone receptor antagonist can be divided into two major classes: growth hormone (GH) analogues and anti-growth hormone receptor (GHR) antibodies. Herein, we tried to explore a new class of growth hormone receptor (GHR) antagonist that may have potential advantages over the traditional antagonists. For this, we developed a monoclonal anti-idiotypic antibody growth hormone, termed CG-86. A series of experiments were conducted to characterize and evaluate this antibody, and the results from a competitive receptor-binding assay, Enzyme Linked Immunosorbent Assays (ELISA) and epitope mapping demonstrate that CG-86 behaved as a typical Ab2β. Next, we examined its antagonistic activity using in vitro cell models, and the results showed that CG-86 could effectively inhibit growth hormone receptor-mediated signalling and effectively inhibit growth hormone-induced Ba/F3-GHR638 proliferation. In summary, these studies show that an anti-idiotypic antibody (CG-86) has promise as a novel growth hormone receptor antagonist. Furthermore, the current findings also suggest that anti-idiotypic antibody may represent a novel strategy to produce a new class of growth hormone receptor antagonist, and this strategy may be applied with other cytokines or growth factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modulatory effect of neuropeptide Y on acetylcholine-induced oedema and vasoconstriction in isolated perfused lungs of rabbit.

PubMed Central

Delaunois, A; Gustin, P; Dessy-Doize, C; Ansay, M

1994-01-01

1. The modulatory role of neuropeptide Y (NPY) on pulmonary oedema induced by acetylcholine and capsaicin was investigated. The effects of NPY on the haemodynamic response to acetylcholine, phenylephrine and substance P were also investigated. 2. Isolated, ventilated, exsanguinated lungs of the rabbit were perfused with a constant flow of recirculating blood-free perfusate. The double/arterial/venous occlusion method was used to partition the total pressure gradient (delta Pt) into four components: the arterial gradient (delta Pa), the pre- and post-capillary gradients (respectively delta Pa' and delta Pv') and the venous pressure gradient (delta Pv). Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). 3. Acetylcholine (10(-8) M to 10(-4) M) and substance P (SP, 10(-10) M to 10(-6) M) induced a concentration-dependent increase in the Kf,c. Capsaicin (10(-4) M) and 5-hydroxytryptamine (5-HT) (10(-4) M) also increased this parameter. NPY (10(-8) M) completely inhibited the effects of acetylcholine and capsaicin on the Kf,c, without preventing the effects of substance P and 5-HT. 4. Acetylcholine induced concentration-dependent vasoconstriction in the precapillary segment. The effect was inhibited by NPY and aspirin, an inhibitor of cyclo-oxygenase, while ketanserin, a 5-HT2 receptor antagonist, and SR140333, a new NK1 antagonist, had no protective effect. Phenylephrine increased delta Pa at high concentration, an effect also inhibited by NPY and aspirin. Substance P had no significant haemodynamic effect. When injected together with NPY, substance P (10(-6) M) induced a significant increase in the total pressure gradient.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 Figure 3 PMID:7532083

[The potential of group II metabotropic glutamate receptor antagonists as a novel antidepressant].

PubMed

Chaki, Shigeyuki

2012-08-01

Recently, abnormalities of glutamatergic transmission have been implicated in the pathophysiology of depression. Moreover, both ketamine, an NMDA receptor antagonist, and riluzole, a modulator of glutamatergic, transmission have been reported to be effective for the treatment of patients with treatment-refractory depression. Based on these findings, extensive studies to develop agents acting on glutamatergic transmission have been conducted. Glutamate receptors are divided into two main subtypes, ionotropic glutamate receptors and metabotropic glutamate (mGlu) receptors, both of which have subtypes. Of these, much attention has been paid to mGlu2/3 receptors. mGlu2/3 receptor antagonists such as MGS0039 and LY341495 have been reported to exert antidepressant effects in animal models of depression including the forced swim test, tail suspension test, learned helplessness paradigm, olfactory bulmectomy model and isolation rearing model, and to enhance serotonin release in the prefrontal cortex and dopamine release in the nucleus accumbens. Moreover, activation of AMPA receptor and mTOR signaling have been suggested to be involved in the antidepressant effects of mGlu2/3 receptor antagonists, as demonstrated in the actions of ketamine. Thus, mGlu2/3 receptor antagonists may share some neural networks with ketamine in exerting their antidepressant effects. In addition, the potential of other agents targeting glutamatergic transmission for novel antidepressants is being investigated.

Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

NASA Astrophysics Data System (ADS)

Dacosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

2015-08-01

α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

Role of the nicotinic acetylcholine receptor α3 subtype in vascular inflammation.

PubMed

Yang, Cui; Li, Zhengtao; Yan, Saimei; He, Yonghui; Dai, Rong; Leung, George Pek-Heng; Pan, Shitian; Yang, Jinyan; Yan, Rong; Du, Guanhua

2016-11-01

Vascular inflammation is a major factor contributing to the development of vascular diseases. The aim of this study was to investigate the role of the nicotinic acetylcholine receptor α3 subtype (α3-nAChR) in vascular inflammation. Vascular inflammation was studied in apolipoprotein E knockout (ApoE -/- ) mice fed a high-fat diet. Inflammatory markers were measured in mouse aortic endothelial cells (MAECs) and macrophages after α3-nAChRs were antagonized pharmacologically, or after the gene of α3-nAChRs was silenced. Treatment with α-conotoxin MII (MII; an α3-nAChR antagonist) increased the number of inflammatory cells infiltrating the aortic walls and further impaired the endothelium-dependent vasodilatations in the aorta of ApoE -/- mice. MII also increased the plasma levels of inflammatory cytokines. Furthermore, the infiltration of classical activated macrophages into the arterial wall of ApoE -/- mice was markedly elevated by MII but that of alternative activated macrophages was reduced. In MAECs, the lipopolysaccharide-stimulated secretion of adhesion molecules and inflammatory cytokines was enhanced by MII, or by silencing the gene of α3-nAChRs. This effect was reversed by inhibitors of the PI3K-Akt-IκKα/β-IκBα-NFκB pathways. In macrophages, the classical activation was enhanced, but the alternative activation was reduced when the gene of α3-nACh receptors was silenced. These effects were prevented by inhibitors of the IκKα/β-IκBα-NFκB and JAK2-STAT6-PPARγ pathways respectively. α3-nAChRs play a pivotal role in regulating the inflammatory responses in endothelial cells and macrophages. The mechanisms involve the modulations of multiple cell signalling pathways. © 2016 The British Pharmacological Society.

Functionalized Congeners of P2Y1 Receptor Antagonists:

DOE Office of Scientific and Technical Information (OSTI.GOV)

de Castro, Sonia; Maruoka, Hiroshi; Hong, Kunlun

2010-01-01

The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of anmore » intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM

In vivo Interactions between α7 Nicotinic Acetylcholine Receptor and Nuclear Peroxisome Proliferator-Activated Receptor-α: Implication for Nicotine Dependence

PubMed Central

Jackson, Asti; Bagdas, Deniz; Muldoon, Pretal P.; Lichtman, Aron H.; Carroll, F. Ivy; Greenwald, Mark; Miles, Michael F.; Damaj, M. Imad

2017-01-01

Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric α7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPARα as a possible mediator of the effect of α7 nAChR activation in nicotine dependence. Our results demonstrate the PPARα antagonist GW6471 blocks actions of the α7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that α7 nAChR activation attenuates nicotine CPP in a PPARα-dependent manner. To evaluate PPARα activation in nicotine dependence we used the selective and potent PPARα agonist, WY-14643 and the clinically used PPARα activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPARα in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPARα plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of α7 nAChRs in nicotine dependence. PMID:28279662

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD).

PubMed

Dwoskin, Linda P; Joyce, B Matthew; Zheng, Guangrong; Neugebauer, Nichole M; Manda, Vamshi K; Lockman, Paul; Papke, Roger L; Bardo, Michael T; Crooks, Peter A

2007-10-15

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N,N'-bis-nicotinium analogs, affording a lead compound, N,N'-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC(50)=500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 microM) inhibited the response to acetylcholine at alpha3beta4, alpha4beta4, alpha4beta2, and alpha1beta1varepsilondelta receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity than choline for the blood-brain barrier choline transporter, suggesting brain bioavailability. TMPD did not inhibit hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.

Zingiberis Siccatum Rhizoma, the active component of the Kampo formula Daikenchuto, induces anti-inflammatory actions through α7 nicotinic acetylcholine receptor activation.

PubMed

Endo, M; Hori, M; Mihara, T; Ozaki, H; Oikawa, T; Odaguchi, H; Hanawa, T

2017-12-01

We previously reported that Daikenchuto (DKT), a gastrointestinal prokinetic Japanese herbal (Kampo) medicine used for the treatment of postoperative ileus (POI), has characteristic potent anti-inflammatory activity. This effect may be partly mediated by the activation of α7 nicotinic acetylcholine receptor (nAChR). In this study, we identified the specific herbs in DKT that induce anti-inflammatory action. The herbal components of DKT were individually administered orally to each mouse four times before and after intestinal manipulation (IM) was carried out on the distal ileum. The anti-inflammatory activity of each crude drug was subsequently evaluated using immunohistochemical analyses of relevant molecules. Treatment with Zingiberis Siccatum Rhizoma (ZSR) but not the other components inhibited the infiltration of cluster of differentiation 68 (CD68)-positive macrophages as effectively as DKT treatment. Selective α7nAChR antagonists, such as methyllycaconitine citrate, or transient receptor potential ankyrin 1 (TRPA1) antagonists, such as HC-030031, significantly inhibited the amelioration of macrophage infiltration by ZSR. The inhibition of macrophage infiltration by ZSR was abolished in both α7nAChR and 5-hydroxytryptamine 4 receptor (5-HT 4 R) knockout mice. Daikenchuto-induced anti-inflammatory activity, which was mediated by inhibiting macrophage infiltration in POI, is dependent on the effects of ZSR. Zingiberis Siccatum Rhizoma activates TRPA1 channels possibly in enterochromaffin (EC) cells to release 5-HT, which stimulates 5-HT 4 R in the myenteric plexus neurons to release ACh, which in turn activates α7nAChR on macrophages to inhibit inflammation in POI. © 2017 John Wiley & Sons Ltd.

Modes of Action, Resistance and Toxicity of Insecticides Targeting Nicotinic Acetylcholine Receptors.

PubMed

Ihara, Makoto; Buckingham, Steven D; Matsuda, Kazuhiko; Sattelle, David B

2017-01-01

Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

BK channel β1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization

PubMed Central

Semenov, Iurii; Wang, Bin; Herlihy, Jeremiah T; Brenner, Robert

2011-01-01

Abstract The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific β1 subunit regulate excitation–contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel β1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK β1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of β1 knockout is eliminated by specific M2 receptor antagonism. The role of BK β1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with β1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK β1 knockout or by paxilline block of BK channels. Normalization of β1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/β1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation–contraction coupling to more negative voltage ranges. PMID:21300746

Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications.

PubMed

Lambrecht, G

2000-11-01

P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.

Synthesis and Nicotinic Acetylcholine Receptor In Vitro and In Vivo Pharmacological Properties of 2'-Fluoro-3'-(substituted phenyl)deschloroepibatidine Analogues of 2'-Fluoro-3'-(4-nitrophenyl)deschloroepibatidine (4-Nitro-PFEB or RTI-7527-102)

PubMed Central

Ondachi, Pauline; Castro, Ana; Luetje, Charles W.; Damaj, M. Imad; Mascarella, S. Wayne; Navarro, Hernán A.; Carroll, F. Ivy

2012-01-01

Herein, we report the synthesis and nicotinic acetylcholine receptor (nAChR) in vitro and in vivo pharmacological properties of 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines 5b–g, analogues of 3'-(4-nitrophenyl) compound 5a. All compounds had high affinity for the α4β2-nAChR and low affinity for α7-nAChR. Initial electrophysiological studies showed that all analogues were antagonists at α4β2-, α3β4-, and α7-nAChRs. The 4-carbamoylphenyl analogue 5g was highly selective for α4β2-nAChR over α3β4- and α7-nAChRs. All the analogues were antagonists of nicotine-induced antinociception in the tail-flick test. Molecular modeling docking studies using agonist-bound form of the X-ray crystal structure of the acetylcholine binding protein suggested several different binding modes for epibatidine, varenicline, and 5a–5g. In particular, a unique binding mode for 5g was suggested by these docking simulations. The high binding affinity, in vitro efficacy, and selectivity of 5g for α4β2-nAChR combined with its nAChR functional antagonist properties suggest that 5g will be a valuable pharmacological tool for studying the nAChR and may have potential as a pharmacotherapy for addiction and other CNS disorders. PMID:22742586

Thyroid Hormone Receptor Antagonists: From Environmental Pollution to Novel Small Molecules.

PubMed

Mackenzie, Louise S

2018-01-01

Thyroid hormone receptors (TRs) are nuclear receptors which control transcription, and thereby have effects in all cells within the body. TRs are an important regulator in many basic physiological processes including development, growth, metabolism, and cardiac function. The hyperthyroid condition results from an over production of thyroid hormones resulting in a continual stimulation of thyroid receptors which is detrimental for the patient. Therapies for hyperthyroidism are available, but there is a need for new small molecules that act as TR antagonists to treat hyperthyroidism. Many compounds exhibit TR antagonism and are considered detrimental to health. Some drugs in the clinic (most importantly, amiodarone) and environmental pollution exhibit TR antagonist properties and thus have the potential to induce hypothyroidism in some people. This chapter provides an overview of novel small molecules that have been specifically designed or screened for their TR antagonist activity as novel treatments for hyperthyroidism. While novel compounds have been identified, to date none have been developed sufficiently to enter clinical trials. Furthermore, a discussion on other sources of TR antagonists is discussed in terms of side effects of current drugs in the clinic as well as environmental pollution. © 2018 Elsevier Inc. All rights reserved.

Change in pharmacological effect of endothelin receptor antagonists in rats with pulmonary hypertension: Role of ETB-receptor expression levels

PubMed Central

Sauvageau, Stéphanie; Thorin, Eric; Villeneuve, Louis; Dupuis, Jocelyn

2013-01-01

Background and purpose The endothelin (ET) system is activated in pulmonary arterial hypertension (PAH). The therapeutic value of pharmacological blockade of ET receptors has been demonstrated in various animal models and led to the current approval and continued development of these drugs for the therapy of human PAH. However, we currently incompletely comprehend what local modifications of this system occur as a consequence of PAH, particularly in small resistance arteries, and how this could affect the pharmacological response to ET receptor antagonists with various selectivities for the receptor subtypes. Therefore, the purposes of this study were to evaluate potential modifications of the pharmacology of the ET system in rat pulmonary resistance arteries from monocrotaline (MCT)-induced pulmonary arterial hypertension. Experimental approach ET-1 levels were quantified by ELISA. PreproET-1, ETA and ETB receptor mRNA expressions were quantified in pulmonary resistance arteries using Q-PCR, while protein expression was evaluated by Western blots. Reactivity to ET-1 of isolated pulmonary resistance arteries was measured in the presence of ETA (A-147627), ETB (A-192621) and dual ETA/B (bosentan) receptor antagonists. Key results In rats with PAH, plasma ET-1 increased (p < 0.001) while pulmonary levels were reduced (p < 0.05). In PAH arteries, preproET-1 (p < 0.05) and ETB receptor (p < 0.001) gene expressions were reduced, as were ETB receptor protein levels (p < 0.05). ET-1 induced similar vasoconstrictions in both groups. In arteries from sham animals, neither bosentan nor the ETA or the ETB receptor antagonists modified the response. In arteries from PAH rats, however, bosentan and the ETA receptor antagonist potently reduced the maximal contraction, while bosentan also reduced sensitivity (p < 0.01). Conclusions and implications The effectiveness of both selective ETA and dual ETA/B receptor antagonists is markedly increased in PAH. Down-regulation of

Models for H₃ receptor antagonist activity of sulfonylurea derivatives.

PubMed

Khatri, Naveen; Madan, A K

2014-03-01

The histamine H₃ receptor has been perceived as an auspicious target for the treatment of various central and peripheral nervous system diseases. In present study, a wide variety of 60 2D and 3D molecular descriptors (MDs) were successfully utilized for the development of models for the prediction of antagonist activity of sulfonylurea derivatives for histamine H₃ receptors. Models were developed through decision tree (DT), random forest (RF) and moving average analysis (MAA). Dragon software version 6.0.28 was employed for calculation of values of diverse MDs of each analogue involved in the data set. The DT classified and correctly predicted the input data with an impressive non-error rate of 94% in the training set and 82.5% during cross validation. RF correctly classified the analogues into active and inactive with a non-error rate of 79.3%. The MAA based models predicted the antagonist histamine H₃ receptor activity with non-error rate up to 90%. Active ranges of the proposed MAA based models not only exhibited high potency but also showed improved safety as indicated by relatively high values of selectivity index. The statistical significance of the models was assessed through sensitivity, specificity, non-error rate, Matthew's correlation coefficient and intercorrelation analysis. Proposed models offer vast potential for providing lead structures for development of potent but safe H₃ receptor antagonist sulfonylurea derivatives. Copyright © 2013 Elsevier Inc. All rights reserved.

Acetylcholine contributes to control the physiological inflammatory response during the peri-implantation period.

PubMed

Paparini, D; Gori, S; Grasso, E; Scordo, W; Calo, G; Pérez Leirós, C; Ramhorst, R; Salamone, G

2015-06-01

Maternal antigen-presenting cells attracted to the pregnant uterus interact with trophoblast cells and modulate their functional profile to favour immunosuppressant responses. Non-neuronal cholinergic system is expressed in human cytotrophoblast cells and in immune cells with homeostatic regulatory functions. The aim of this work was to evaluate whether non-neuronal acetylcholine conditions maternal monocyte and DC migration and activation profiles. We used an in vitro model resembling maternal-placental interface represented by the co-culture of human trophoblast cells (Swan-71 cell line) and monocytes or DC. When cytotrophoblast cells were treated with neostigmine (Neo) to concentrate endogenous acetylcholine levels, monocyte migration was increased. In parallel, high levels of IL-10 and decreased levels of TNF-α were observed upon interaction of maternal monocytes with trophoblast cells. This effect was synergized by Neo and was prevented by atropine, a muscarinic acetylcholine receptor antagonist. Similarly, trophoblast cells increased the migration of DC independently of Neo treatment; however, enhanced IL-10 and MCP-1 synthesis in trophoblast-DC co-cultures with no changes in TNF-α and IL-6 was observed. In fact, there were no changes in HLA-DR, CD86 or CD83 expression. Finally, trophoblast cells treated with Neo increased the expression of two antigen-presenting cells attracting chemokines, MCP-1, MIP-1α and RANTES through muscarinic receptors, and it was prevented by atropine. Our present results support a novel role of acetylcholine synthesized by trophoblast cells to modulate antigen-presenting cell migration and activation favouring an immunosuppressant profile that contributes to immune homeostasis maintenance at the maternal-foetal interface. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Behavioral, biological, and chemical perspectives on targeting CRF1 receptor antagonists to treat alcoholism

PubMed Central

Zorrilla, Eric P.; Heilig, Markus; de Wit, Harriet; Shaham, Yavin

2013-01-01

Background Alcohol use disorders are chronic disabling conditions for which existing pharmacotherapies have only modest efficacy. In the present review, derived from the 2012 Behavior, Biology and Chemistry “Translational Research in Addiction” symposium, we summarize the anti-relapse potential of corticotropin-releasing factor type 1 (CRF1) receptor antagonists to reduce negative emotional symptoms of acute and protracted alcohol withdrawal and stress-induced relapse to alcohol seeking. Methods We review the biology of CRF1 systems, the activity of CRF1 receptor antagonists in animal models of anxiolytic and antidepressant activity, and experimental findings in alcohol addiction models. We also update the clinical trial status of CRF1 receptor antagonists, including pexacerfont (BMS-562086), emicerfont (GW876008), verucerfont (GSK561679), CP316311, SSR125543A, R121919/NBI30775, R317573/19567470/CRA5626, and ONO-2333Ms. Finally, we discuss the potential heterogeneity and pharmacogenomics of CRF1 receptor pharmacotherapy for alcohol dependence. Results The evidence suggests that brain penetrant-CRF1 receptor antagonists have therapeutic potential for alcohol dependence. Lead compounds with clinically desirable pharmacokinetic properties now exist, and longer receptor residence rates (i.e., slow dissociation) may predict greater CRF1 receptor antagonist efficacy. Functional variants in genes that encode CRF system molecules, including polymorphisms in Crhr1 (rs110402, rs1876831, rs242938) and Crhbp genes (rs10055255, rs3811939) may promote alcohol seeking and consumption by altering basal or stress-induced CRF system activation. Conclusions Ongoing clinical trials with pexacerfont and verucerfont in moderately to highly severe dependent anxious alcoholics may yield insight as to the role of CRF1 receptor antagonists in a personalized medicine approach to treat drug or alcohol dependence. PMID:23294766

Discovery of Tertiary Sulfonamides as Potent Liver X Receptor Antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zuercher, William J.; Buckholz†, Richard G.; Campobasso, Nino

2010-08-12

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

PubMed

Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

2010-04-22

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Progesterone receptor isoforms, agonists and antagonists differentially reprogram estrogen signaling

PubMed Central

Singhal, Hari; Greene, Marianne E.; Zarnke, Allison L.; Laine, Muriel; Al Abosy, Rose; Chang, Ya-Fang; Dembo, Anna G.; Schoenfelt, Kelly; Vadhi, Raga; Qiu, Xintao; Rao, Prakash; Santhamma, Bindu; Nair, Hareesh B.; Nickisch, Klaus J.; Long, Henry W.; Becker, Lev; Brown, Myles; Greene, Geoffrey L.

2018-01-01

Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct but overlapping genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. The better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher in vivo anti-tumor activity of therapies that combine tamoxifen with PR antagonists and modulators. This study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist), from unique isoform and ligand-specific effects will inform the development of biomarkers for patient selection and translation of PR

Discovery of an Orally Bioavailable Gonadotropin-Releasing Hormone Receptor Antagonist.

PubMed

Kim, Seon-Mi; Lee, Minhee; Lee, So Young; Park, Euisun; Lee, Soo-Min; Kim, Eun Jeong; Han, Min Young; Yoo, Taekyung; Ann, Jihyae; Yoon, Suyoung; Lee, Jiyoun; Lee, Jeewoo

2016-10-13

We developed a compound library for orally available gonadotropin-releasing hormone (GnRH) receptor antagonists that were based on a uracil scaffold. On the basis of in vitro activity and CYP inhibition profile, we selected 18a (SKI2496) for further in vivo studies. Compound 18a exhibited more selective antagonistic activity toward the human GnRH receptors over the GnRHRs in monkeys and rats, and this compound also showed inhibitory effects on GnRH-mediated signaling pathways. Pharmacokinetic and pharmacodynamic evaluations of 18a revealed improved bioavailability and superior gonadotropic suppression activity compared with Elagolix, the most clinically advanced compound. Considering that 18a exhibited highly potent and selective antagonistic activity toward the hGnRHRs along with favorable pharmacokinetic profiles, we believe that 18a may represent a promising candidate for an orally available hormonal therapy.

Eplerenone: a selective aldosterone receptor antagonist for patients with heart failure.

PubMed

Barnes, Brian J; Howard, Patricia A

2005-01-01

To evaluate the pharmacology, pharmacokinetics, safety, and clinical use of eplerenone in heart failure (HF). English-language MEDLINE searches were performed from 1966 to May 2004. Key words included eplerenone, aldosterone receptor antagonist, heart failure, myocardial infarction, left-ventricular dysfunction, and cost-effectiveness. Additional references were identified from bibliographies of selected articles. Human trials evaluating the efficacy, safety, and cost-effectiveness of aldosterone receptor antagonists in HF were evaluated. Eplerenone is the first selective aldosterone receptor antagonist. The drug is indicated to improve the survival of stable patients with left-ventricular systolic dysfunction (ejection fraction <40%) and clinical evidence of HF following acute myocardial infarction. Efficacy and safety in this population have been demonstrated in a large, randomized clinical trial. Eplerenone is associated with severe and sometimes life-threatening hyperkalemia. Patients with reduced renal function and diabetes, as well as those on other drugs that increase potassium levels, are at highest risk. Eplerenone is metabolized by the cytochrome P450 system and may interact with drugs that interfere with this system. A major advantage of eplerenone over the nonselective aldosterone receptor antagonist spironolactone is lack of binding to progesterone and androgen receptors, which is associated with drug-induced gynecomastia, breast pain, and impotence. The addition of eplerenone to traditional HF therapy has been shown to reduce morbidity and mortality in patients who develop left-ventricular dysfunction after acute myocardial infarction. Eplerenone's selectivity reduces sex hormone-related adverse effects. Despite these benefits, the overall cost-effectiveness has yet to be determined.

Activation of m1 muscarinic acetylcholine receptor induces surface transport of KCNQ channels through a CRMP-2-mediated pathway.

PubMed

Jiang, Ling; Kosenko, Anastasia; Yu, Clinton; Huang, Lan; Li, Xuejun; Hoshi, Naoto

2015-11-15

Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway. © 2015. Published by The Company of Biologists Ltd.

Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

PubMed

Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

2008-10-01

GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

PubMed Central

Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

2013-01-01

Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice. [Ethanol effects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watson, M.; Ming, X.; McArdle, J.J.

Differences in voluntary ethanol consumption have been noted in various inbred strains of mice and pharmacogenetic approaches have been used to study the mechanisms of action of many drugs such as ethanol. Long-sleep (LS) and short-sleep (SS) mice, selectively bred for differences in ethanol induced narcosis, provide a method by which a relationship between the differential responsiveness of these geno-types and muscarinic acetylcholine receptors (mAChR) may be evaluated. Sleep times after injection of 3ml ethanol/kg (i.p.) verified the higher sensitivity of LS vs. SS. Mean body weights of LS (26.5g) vs. SS (22g) were also significantly (p<.01) greater. Binding assaysmore » for ({sup 3}H)(-) quinuclidinylbenzilate (({sup 3}H)(-)QNB), a specific but nonsubtype selective mAChR antagonist, ({sup 3}H)pirenzepine (({sup 3}H)PZ), a specific M1 mAChR antagonist and ({sup 3}H)11-2-((2-((diethylamino) methyl)-1-piperidinyl) acetyl)-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, (({sup 3}H)AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (K{sub d}) and density (B{sub max}) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) ({sup 3}H)(-)QNB binding suggests that SS have fewer mAChR's than LS in many areas. These differences may relate to their differential ethanol sensitivity.« less

Nonpeptidic angiotensin II AT₁ receptor antagonists derived from 6-substituted aminocarbonyl and acylamino benzimidazoles.

PubMed

Zhang, Jun; Wang, Jin-Liang; Yu, Wei-Fa; Zhou, Zhi-Ming; Tao, Wen-Chang; Wang, Yi-Cheng; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

2013-11-01

Both 6-substituted aminocarbonyl and acylamino benzimidazole derivatives were designed and synthesized as nonpeptidic angiotensin II AT₁ receptor antagonists. Compounds 6f, 6g, 11e, 11f, 11g, and 12 showed nanomolar AT₁ receptor binding affinity and high AT₁ receptor selectivity over AT₂ receptor in a preliminary pharmacological evaluation. Among them, the two most active compounds 6f (AT₁ IC₅₀ = 3 nM, AT₂ IC₅₀ > 10,000 nM, PA₂ = 8.51) and 11g (AT₁ IC₅₀ = 0.1 nM, AT₂ IC₅₀ = 149 nM, PA₂ = 8.43) exhibited good antagonistic activity in isolated rabbit aortic strip functional assay. In addition, they were orally active AT₁ receptor antagonists in spontaneous hypertensive rats. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Visualization and functional testing of acetylcholine receptor-like molecules in cochlear outer hair cells.

PubMed

Plinkert, P K; Gitter, A H; Zimmermann, U; Kirchner, T; Tzartos, S; Zenner, H P

1990-02-01

The efferent nerve endings at outer hair cells (OHCs) have been suggested to regulate active mechanical processes in the cochlea. The discovery of acetylcholine (ACh)-producing and -degrading enzymes in these synapses gave rise to the speculation that ACh might be one of the efferent transmitters. However, there has as yet been no identification and characterization of any corresponding receptor in OHCs which is required for further clarification of this question. In the present paper existence, location and first characterization of acetylcholine receptors (AChRs) in OHCs are reported. Using two anti-AChR monoclonal antibodies, AChR epitopes were found forming a cup at the basal end of the OHCs opposite to the efferent nerve endings. Furthermore, the studied molecules could be shown to extend through the cell membrane. In addition, the denervated OHC AChR-epitopes seem to move by lateral diffusion. Application of Carbachol and ACh to the basal pole of OHCs induced a weak, reversible cell contraction. Pharmacological controls revealed, that hte motile responses were mediated by the AChRs.

MOLECULAR PROBES FOR MUSCARINIC RECEPTORS: FUNCTIONALIZED CONGENERS OF SELECTIVE MUSCARINIC ANTAGONISTS

PubMed Central

Jacobson, Kenneth A.; Fischer, Bilha; van Rhee, A. Michiel

2012-01-01

Summary The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of TAC (telenzepine amine congener), an n-decylamino derivative of the ml-selective antagonist, with the fluorescent trisulfonated pyrene dye Cascade Blue may be useful for assaying the receptor as an alternative to radiotracers. In a rat m3 receptor mutant containing a single amino acid substitution in the sixth transmembrane domain (Asn507 to Ala) the parent telenzepine lost 636-fold in affinity, while TAC lost only 27-fold. Thus, the decylamino group of TAC stabilizes the bound state and thus enhances potency by acting as a distal anchor in the receptor binding site. We have built a computer-assisted molecular model of the transmembrane regions of muscarinic receptors based on homology with the G-protein coupled receptor rhodopsin, for which a low resolution structure is known. We have coordinated the antagonist pharmacophore (tricyclic and piperazine moieties) with residues of the third and seventh helices of the rat m3 receptor. Although the decylamino chain of TAC is likely to be highly flexible and may adopt many conformations, we located one possible site for a salt bridge formation with the positively charged −NH3+ group, i.e. Asp113 in helix II. PMID:10188781

Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

USGS Publications Warehouse

Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

2014-01-01

Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

Effect of thromboxane antagonists on ozone-induced airway responses in dogs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jones, G.L.; Lane, C.G.; O'Byrne, P.M.

1990-09-01

Airway hyperresponsiveness after inhaled ozone in dogs may occur as a result of thromboxane release in the airway. In this study, two thromboxane receptor antagonists, L-655,240 and L-670,596, were used in doses that inhibit the response to an inhaled thromboxane mimetic, U-46619, to determine further the role of thromboxane in ozone-induced airway hyperresponsiveness. Dogs were studied on 2 days separated by 1 wk. On each day, the dogs inhaled ozone (3 ppm) for 30 min. On one randomly assigned day, 10 dogs received an infusion of L-655,240 (5 mg.kg-1.h-1) and 5 dogs received an infusion of L-670,596 (1 mg.kg-1.h-1); onmore » the other day dogs received a control infusion. Airway responses to doubling doses of acetylcholine were measured before and after inhalation of ozone and were expressed as the concentration of acetylcholine giving a rise in resistance of 5 cmH2O.l-1.s from baseline (acetylcholine provocation concentration). The development of airway hyperresponsiveness after ozone was not inhibited by the thromboxane antagonists. The mean log difference in the acetylcholine provocative concentration before and after ozone on the L-655,240 treatment day was 0.62 +/- 0.12 (SE) and on the control day was 0.71 +/- 0.12 (P = 0.48); on the L-670,596 treatment day the mean log difference was 0.68 +/- 0.15 (SE) and on the control day it was 0.75 +/- 0.19 (P = 0.45). These results do not support an important role for thromboxane in causing ozone-induced airway hyperresponsiveness.« less

OPC-21268, an orally effective, nonpeptide vasopressin V1 receptor antagonist.

PubMed

Yamamura, Y; Ogawa, H; Chihara, T; Kondo, K; Onogawa, T; Nakamura, S; Mori, T; Tominaga, M; Yabuuchi, Y

1991-04-26

An orally effective, nonpeptide, vasopressin V1 receptor antagonist, OPC-21268, has been identified. This compound selectively antagonized binding to the V1 subtype of the vasopressin receptor in a competitive manner. In vivo, the compound acted as a specific antagonist of arginine vasopressin (AVP)-induced vasoconstriction. After oral administration in conscious rats, the compound also antagonized pressor responses to AVP. OPC-21268 can be used to study the physiological role of AVP and may be therapeutically useful in the treatment of hypertension and congestive heart failure.

In vivo interactions between α7 nicotinic acetylcholine receptor and nuclear peroxisome proliferator-activated receptor-α: Implication for nicotine dependence.

PubMed

Jackson, Asti; Bagdas, Deniz; Muldoon, Pretal P; Lichtman, Aron H; Carroll, F Ivy; Greenwald, Mark; Miles, Michael F; Damaj, M Imad

2017-05-15

Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric α7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPARα as a possible mediator of the effect of α7 nAChR activation in nicotine dependence. Our results demonstrate the PPARα antagonist GW6471 blocks actions of the α7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that α7 nAChR activation attenuates nicotine CPP in a PPARα-dependent manner. To evaluate PPARα activation in nicotine dependence we used the selective and potent PPARα agonist, WY-14643 and the clinically used PPARα activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPARα in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPARα plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of α7 nAChRs in nicotine dependence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of M2 and M3 muscarinic acetylcholine receptor subtypes in activation of bladder afferent pathways in spinal cord injured rats.

PubMed

Matsumoto, Yoshihiro; Miyazato, Minoru; Yokoyama, Hitoshi; Kita, Masafumi; Hirao, Yoshihiko; Chancellor, Michael B; Yoshimura, Naoki

2012-05-01

To evaluate the role of M2 and M3 muscarinic acetylcholine receptor (mAChR) subtypes in the activation of bladder afferent pathways in rats with chronic spinal cord injury (SCI). Adult female Sprague-Dawley rats were spinalized at the T9 level. Continuous cystometry was performed under awake conditions 2 or 4 weeks after SCI. The effects of intravesical administration of an mAChR agonist (oxotremorine-methiodide), a nonselective antagonist (atropine), an M2-selective antagonist (methoctramine), and an M3-selective antagonist (darifenacin) were examined. After cystometry, the bladder was removed and separated into the mucosa and detrusor, and the M2 and M3 mAChR mRNA expression in the mucosa was determined using real-time quantitative polymerase chain reaction. At 2 and 4 weeks after SCI, intravesical administration of a nonselective mAChR agonist (25 μM oxotremorine-methiodide) increased the area under the curve of nonvoiding contractions, although the intercontraction interval of voiding contractions and maximal voiding pressure did not change. This effect was blocked by atropine and methoctramine (10 μM) but not by darifenacin (50 μM). However, mAChR antagonists alone (10-50 μM) had no effect on cystometric parameters. M2 mAChR mRNA expression was increased in the mucosa of SCI rats compared with that in normal rats. Our results suggest that the M2 mAChR subtype plays an important role in bladder afferent activation that enhances detrusor overactivity in SCI rats. However, because mAChR antagonists alone did not affect any cystometric parameters, the muscarinic mechanism controlling bladder afferent activity might not be involved in the emergence of detrusor overactivity in SCI. Copyright © 2012 Elsevier Inc. All rights reserved.

Peptides from puff adder Bitis arietans venom, novel inhibitors of nicotinic acetylcholine receptors.

PubMed

Vulfius, Catherine A; Spirova, Ekaterina N; Serebryakova, Marina V; Shelukhina, Irina V; Kudryavtsev, Denis S; Kryukova, Elena V; Starkov, Vladislav G; Kopylova, Nina V; Zhmak, Maxim N; Ivanov, Igor A; Kudryashova, Ksenia S; Andreeva, Tatyana V; Tsetlin, Victor I; Utkin, Yuri N

2016-10-01

Phospholipase A 2 (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bitis arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3-10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC 50 of about 50 μM and 250 μM, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC 50 of about 3 μM. The peptides did not compete with radioactive α-bungarotoxin for binding to Torpedo and α7 nAChRs at concentration up to 200 μM that suggests non-competitive mode of inhibition. Calcium imaging studies on α7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on α7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC 50 of 20.6 ± 3.93 μM. On both α7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 μM, the value being close to IC 50 on α7 nAChR. These data are

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Resende, R.R.; Alves, A.S.; Britto, L.R.G

2008-04-15

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereasmore » intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.« less

a2* Nicotinic Acetylcholine Receptors Influence Hippocampus-Dependent Learning and Memory in Adolescent Mice

ERIC Educational Resources Information Center

Lotfipour, Shahrdad; Mojica, Celina; Nakauchi, Sakura; Lipovsek, Marcela; Silverstein, Sarah; Cushman, Jesse; Tirtorahardjo, James; Poulos, Andrew; Elgoyhen, Ana Belén; Sumikawa, Katumi; Fanselow, Michael S.; Boulter, Jim

2017-01-01

The absence of a2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of a2* nAChRs ("Chrna2"[superscript L9'S/L9'S] and…

Greater Ethanol Inhibition of Presynaptic Dopamine Release in C57BL/6J than DBA/2J Mice: Role of Nicotinic Acetylcholine Receptors

PubMed Central

Yorgason, Jordan T.; Rose, Jamie H.; McIntosh, J. Michael; Ferris, Mark J.; Jones, Sara R.

2014-01-01

The mesolimbic dopamine system, originating in the ventral tegmental area (VTA) and projecting to the nucleus accumbens (NAc), has been heavily implicated in the reinforcing effects of ethanol. Recent slice voltammetry studies have shown that ethanol inhibits dopamine release selectively during highfrequency activity that elicits phasic dopamine release shown to be important for learning and reinforcement. Presently, we examined ethanol inhibition of electrically evoked NAc dopamine in two mouse strains with divergent dopamine responses to ethanol, C57BL/6 (C57) and DBA/2J (DBA) mice. Previous electrophysiology and microdialysis studies have demonstrated greater ethanol induced VTA dopaminergic firing and NAc dopamine elevations in DBA compared to C57 mice. Additionally, DBA mice have greater ethanol responses in dopamine-related behaviors, including hyperlocomotion and conditioned place preference. Currently, we demonstrate greater sensitivity of ethanol inhibition of NAc dopamine signaling in C57 compared to DBA mice. The reduced sensitivity to ethanol inhibition in DBA mice may contribute to the overall greater ethanol-induced dopamine signaling and related behaviors observed in this strain. NAc cholinergic activity is known to potently modulate terminal dopamine release. Additionally, ethanol is known to interact with multiple aspects of nicotinic acetylcholine receptor activity. Therefore, we examined ethanol-mediated inhibition of dopamine release at two ethanol concentrations (80 and 160mM) during bath application of the non-selective nicotinic receptor antagonist mecamylamine, as well as compounds selective for the β2- (DhβE) and α6- (α-conotoxin MII [H9A; L15A]) subunit-containing receptors. Mecamylamine and DhβE decreased dopamine release and reduced ethanol's inhibitory effects on dopamine in both DBA and C57 mice. Further, α-conotoxin also reduced the dopamine release and the dopamine-inhibiting effects of ethanol at the 80mM, but not 160m

Nicotinic acetylcholine receptor ligands; a patent review (2006-2011)

PubMed Central

Gündisch, Daniela; Eibl, Christoph

2012-01-01

Introduction Nicotinic acetylcholine receptors (nAChRs), pentameric ligand-gated cation channels, are potential targets for the development of therapeutics for a variety of disease states. Areas covered This article is reviewing recent advances in the development of small molecule ligands for diverse nAChR subtypes and is a continuation of an earlier review in this journal. Expert opinion The development of nAChR ligands with preference for α4β2 or α7 subtypes for the treatment of CNS disorders are in the most advanced developmental stage. In addition, there is a fast growing interest to generate so-called PAMs, positive allosteric modulators, to influence the channels’ functionalities. PMID:22098319

Widespread Decrease of Nicotinic Acetylcholine Receptors in Parkinson's Disease

PubMed Central

Ichise, Masanori; Zoghbi, Sami S; Liow, Jeih-San; Ghose, Subroto; Vines, Douglass C; Sangare, Janet; Lu, Jian-Qiang; Cropley, Vanessa L; Iida, Hidehiro; Kim, Kyeong Min; Cohen, Robert M; Bara-Jimenez, William; Ravina, Bernard; Innis, Robert B

2005-01-01

Nicotinic acetylcholine receptors (nAChRs) have close interactions with the dopaminergic system and play critical roles in cognitive function. nAChRs were imaged in 10 non-demented Parkinson's disease (PD) patients and 15 age-matched healthy subjects using a single photon emission computed tomography ligand [123I]5-iodo-3-[2(S)-2-azetidinylmethoxy]pyridine. Using an arterial input function, we measured the total distribution volume (V; specific plus non-displaceable) as well as the delivery (K1). PD showed a widespread significant decrease (∼10%) of V in both cortical and subcortical regions without a significant change in K1. These results indicate the importance of extending the study to demented patients. PMID:16374823

Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lentz, T.L.

1991-11-12

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similarmore » segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.« less

Characterization of a novel non-peptide vasopressin V1 receptor antagonist (OPC-21268) in the rat.

PubMed

Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I

1993-08-01

A non-peptide, orally effective, vasopressin (AVP) V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl)-3,4-dihydro-2(1H)-quinolinone (OPC-21268) has recently been described. This paper reports the in-vitro and in-vivo characterization of OPC-21268 binding to vasopressin receptors in rat liver and kidney. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, 125I-labelled [d(CH2)5,sarcosine7]AVP to V1 receptors in both rat liver and kidney medulla membranes. The concentration of OPC-21268 that displaced 50% of specific AVP binding (IC50) was 40 +/- 3 nmol/l for liver V1 and 15 +/- 2 nmol/l for kidney V1 receptors (mean +/- S.E.M.; n = 3). OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)]d(CH2)5,D-Ile2,Ile4] AVP binding to V2 receptors in renal medulla membranes (IC50 > 0.1 mmol/l). After oral administration to rats, OPC-21268 was an effective V1 antagonist in a time- and dose-dependent manner. Binding kinetic studies showed that OPC-21268 acted as a competitive antagonist at the liver V1 receptor in vitro and in vivo, in addition to its in-vitro competitive effects at the renal V1 receptor. OPC-21268 shows promise as an orally active V1 antagonist.

Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists.

PubMed

Rivera, Alicia

2007-09-01

Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg.kg(-1).day(-1) intraperitoneally for 14 days), an ET-1 receptor B (ET(B)) antagonist, induced a significant decrease in Gardos channel activity (1.7 +/- 0.1 to 1.0 +/- 0.4 mmol.10(13) cell(-1).h(-1), n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D(50); n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ET(A)) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ET(B)/ET(A) antagonists but not by ET(A) antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ET(B) receptor, leading to Gardos channel modulation in SCD.

Combining the α1-Adrenergic Receptor Antagonist, Prazosin, with the β-Adrenergic Receptor Antagonist, Propranolol, Reduces Alcohol Drinking More Effectively Than Either Drug Alone

PubMed Central

Rasmussen, Dennis D; Beckwith, Lauren E; Kincaid, Carrie L; Froehlich, Janice C

2014-01-01

Background Evidence suggests that activation of the noradrenergic system may contribute to alcohol drinking in animals and humans. Our previous studies demonstrated that blocking α1-adrenergic receptors with the antagonist, prazosin, decreased alcohol drinking in rats under various conditions. Since noradrenergic activation is also regulated by β-adrenergic receptors, we now examine the effects of the β-adrenergic receptor antagonist, propranolol, alone or in combination with prazosin, on alcohol drinking in rats selectively bred for high voluntary alcohol intake and alcohol preference (P line). Methods Two studies were conducted with male P rats. In study one, rats were allowed to become alcohol-dependent during 14 weeks of ad libitum access to food, water and 20% alcohol and the effect of propranolol (5–15 mg/kg, IP) and prazosin (1–2 mg/kg, IP) on alcohol intake during withdrawal were assessed. In study two, the effect of propranolol (5 mg/kg, IP) and prazosin (2 mg/kg, IP) on alcohol intake following prolonged imposed abstinence was assessed. Results Alcohol drinking following propranolol treatment was variable, but the combination of propranolol + prazosin consistently suppressed alcohol drinking during both alcohol withdrawal and following prolonged imposed abstinence, and the combination of these two drugs was more effective than was treatment with either drug alone. Conclusions Treatment with prazosin + propranolol, or a combination of other centrally active α1- and β-adrenergic receptor antagonists, may assist in preventing alcohol relapse in some individuals. PMID:24891220

Mixed nicotinic-muscarinic properties of the alpha9 nicotinic cholinergic receptor.

PubMed

Verbitsky, M; Rothlin, C V; Katz, E; Elgoyhen, A B

2000-10-01

The rat alpha9 nicotinic acetylcholine receptor (nAChR) was expressed in Xenopus laevis oocytes and tested for its sensitivity to a wide variety of cholinergic compounds. Acetylcholine (ACh), carbachol, choline and methylcarbachol elicited agonist-evoked currents, giving maximal or near maximal responses. Both the nicotinic agonist suberyldicholine as well as the muscarinic agonists McN-A-343 and methylfurtrethonium behaved as weak partial agonists of the receptor. Most classical cholinergic compounds tested, being either nicotinic (nicotine, epibatidine, cytisine, methyllycaconitine, mecamylamine, dihydro-beta-erythroidine), or muscarinic (muscarine, atropine, gallamine, pilocarpine, bethanechol) agonists and antagonists, blocked the recombinant alpha9 receptor. Block by nicotine, epibatidine, cytisine, methyllycaconitine and atropine was overcome at high ACh concentrations, suggesting a competitive type of block. The present results indicate that alpha9 displays mixed nicotinic-muscarinic features that resemble the ones described for the cholinergic receptor of cochlear outer hair cells (OHCs). We suggest that alpha9 contains the structural determinants responsible for the pharmacological properties of the native receptor.

The Vagal Nerve Stimulates Activation of the Hepatic Progenitor Cell Compartment via Muscarinic Acetylcholine Receptor Type 3

PubMed Central

Cassiman, David; Libbrecht, Louis; Sinelli, Nicoletta; Desmet, Valeer; Denef, Carl; Roskams, Tania

2002-01-01

In the rat the hepatic branch of the nervus vagus stimulates proliferation of hepatocytes after partial hepatectomy and growth of bile duct epithelial cells after bile duct ligation. We studied the effect of hepatic vagotomy on the activation of the hepatic progenitor cell compartment in human and rat liver. The number of hepatic progenitor cells and atypical reactive ductular cells in transplanted (denervated) human livers with hepatitis was significantly lower than in innervated matched control livers and the number of oval cells in vagotomized rat livers with galactosamine hepatitis was significantly lower than in livers of sham-operated rats with galactosamine hepatitis. The expression of muscarinic acetylcholine receptors (M1-M5 receptor) was studied by immunohistochemistry and reverse transcriptase-polymerase chain reaction. In human liver, immunoreactivity for M3 receptor was observed in hepatic progenitor cells, atypical reactive ductules, intermediate hepatocyte-like cells, and bile duct epithelial cells. mRNA for the M1-M3 and the M5 receptor, but not the M4 receptor, was detected in human liver homogenates. In conclusion, the hepatic vagus branch stimulates activation of the hepatic progenitor cell compartment in diseased liver, most likely through binding of acetylcholine to the M3 receptor expressed on these cells. These findings may be of clinical importance for patients with a transplant liver. PMID:12163377

Tranylcypromine Substituted cis-Hydroxycyclobutylnaphthamides as Potent and Selective Dopamine D3 Receptor Antagonists

PubMed Central

2015-01-01

We report a class of potent and selective dopamine D3 receptor antagonists based upon tranylcypromine. Although tranylcypromine has a low affinity for the rat D3 receptor (Ki = 12.8 μM), our efforts have yielded (1R,2S)-11 (CJ-1882), which has Ki values of 2.7 and 2.8 nM at the rat and human dopamine D3 receptors, respectively, and displays respective selectivities of >10000-fold and 223-fold over the rat and human D2 receptors. Evaluation in a β-arrestin functional assay showed that (1R,2S)-11 is a potent and competitive antagonist at the human D3 receptor. PMID:24848155

(D-Phe/sup 12/)bombesin analogues: a new class of bombesin receptor antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heinz-Erian, P.; Coy, D.H.; Tamura, M.

1987-03-01

Previous attempts to develop analogues of bombesin that function as specific receptor antagonists have been unsuccessful. Alteration of the histidine in luteinizing hormone releasing factor has resulted in analogues that function as competitive antagonists. In the present study the authors have used a similar strategy and altered the histidine in bombesin. (D-Phe/sup 12/)bombesin, (D-Phe/sup 12/,Leu/sup 14/)bombesin, and (Try/sup 4/, D-)je/sup 12/) bombesin did not stimulate amylase release from guinea pig pancreatic acini when present alone, but each analog inhibited bombesin-stimulated secretion. For each analog, detectable inhibition occurred at 1 ..mu..M and half-maximal inhibition at 4 ..mu..M. Each analog inhibited amylasemore » release by bombesin and other agonists that stimulate secretion by interacting with bombesin receptors. The analogues of bombesin did not alter stimulation by substance P or other agonists that interact with other receptors. The inhibition of the action of bombesin was competitive with Schild plots having slopes of 1.0. Each analog also inhibited binding of /sup 125/I-labeled (Try/sup 4/) bombesin but not /sup 125/I-labeled substance P. These results demonstrate that (D-Phe/sup 12/) analogues of bombesin function as bombesin receptor antagonists and are the only bombesin receptor antagonists that interact only with the bombesin receptor. Because of their specificity, these analogues may prove useful for defining the role of bombesin in various physiological or pathological processes.« less

MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist.

PubMed

Cialdai, Cecilia; Tramontana, Manuela; Patacchini, Riccardo; Lecci, Alessandro; Catalani, Claudio; Catalioto, Rose-Marie; Meini, Stefania; Valenti, Claudio; Altamura, Maria; Giuliani, Sandro; Maggi, Carlo Alberto

2006-11-07

The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2receptor agonist, [betaAla8]neurokinin A(4-10) (3 nmol/kg i.v.), either after intravenous (ED50 0.18 micromol/kg), intraduodenal (ED50 3.16 micromol/kg) or oral administration (10-30 micromol/kg) without affecting, at 3 micromol/kg, i.v., the colonic contractions produced by the NK1 receptor selective agonist [Sar9]substance P sulfone (3 nmol/kg i.v.). In addition MEN15596 was effective in inhibiting bronchoconstriction produced by i.v. administration of [betaAla8]neurokinin A(4-10). Overall the results indicate that MEN15596 is a potent and selective

Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.

2006-06-23

{alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

An autoinflammatory disease with deficiency of the interleukin-1-receptor antagonist.

PubMed

Aksentijevich, Ivona; Masters, Seth L; Ferguson, Polly J; Dancey, Paul; Frenkel, Joost; van Royen-Kerkhoff, Annet; Laxer, Ron; Tedgård, Ulf; Cowen, Edward W; Pham, Tuyet-Hang; Booty, Matthew; Estes, Jacob D; Sandler, Netanya G; Plass, Nicole; Stone, Deborah L; Turner, Maria L; Hill, Suvimol; Butman, John A; Schneider, Rayfel; Babyn, Paul; El-Shanti, Hatem I; Pope, Elena; Barron, Karyl; Bing, Xinyu; Laurence, Arian; Lee, Chyi-Chia R; Chapelle, Dawn; Clarke, Gillian I; Ohson, Kamal; Nicholson, Marc; Gadina, Massimo; Yang, Barbara; Korman, Benjamin D; Gregersen, Peter K; van Hagen, P Martin; Hak, A Elisabeth; Huizing, Marjan; Rahman, Proton; Douek, Daniel C; Remmers, Elaine F; Kastner, Daniel L; Goldbach-Mansky, Raphaela

2009-06-04

Autoinflammatory diseases manifest inflammation without evidence of infection, high-titer autoantibodies, or autoreactive T cells. We report a disorder caused by mutations of IL1RN, which encodes the interleukin-1-receptor antagonist, with prominent involvement of skin and bone. We studied nine children from six families who had neonatal onset of sterile multifocal osteomyelitis, periostitis, and pustulosis. Response to empirical treatment with the recombinant interleukin-1-receptor antagonist anakinra in the first patient prompted us to test for the presence of mutations and changes in proteins and their function in interleukin-1-pathway genes including IL1RN. We identified homozygous mutations of IL1RN in nine affected children, from one family from Newfoundland, Canada, three families from The Netherlands, and one consanguineous family from Lebanon. A nonconsanguineous patient from Puerto Rico was homozygous for a genomic deletion that includes IL1RN and five other interleukin-1-family members. At least three of the mutations are founder mutations; heterozygous carriers were asymptomatic, with no cytokine abnormalities in vitro. The IL1RN mutations resulted in a truncated protein that is not secreted, thereby rendering cells hyperresponsive to interleukin-1beta stimulation. Patients treated with anakinra responded rapidly. We propose the term deficiency of the interleukin-1-receptor antagonist, or DIRA, to denote this autosomal recessive autoinflammatory disease caused by mutations affecting IL1RN. The absence of interleukin-1-receptor antagonist allows unopposed action of interleukin-1, resulting in life-threatening systemic inflammation with skin and bone involvement. (ClinicalTrials.gov number, NCT00059748.) 2009 Massachusetts Medical Society

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists.

PubMed

Moody, Terry W; Tashakkori, Nicole; Mantey, Samuel A; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB 2 R), neuromedin B receptor (BB 1 R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB 1 R, BB 2 R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB 1 R, BB 2 R, and BRS-3 with similar affinity ( K i = 1.4-10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca 2+ in human lung cancer cells transfected with BB 1 R, BB 2 R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists.

Effects of tachykinin receptor agonists and antagonists on the guinea-pig isolated oesophagus.

PubMed

Kerr, K P

2000-11-01

1. Vagal nerve stimulation of the guinea-pig isolated oesophagus produced a triphasic tetrodotoxin (TTX)-sensitive contractile response. The third phase, which was resistant to ganglion blocking drugs, was selectively abolished by capsaicin, suggesting the involvement of one or more neuropeptides released from afferent neurons. Receptors on cholinergic neurons were subsequently activated because the response was atropine sensitive. Contractile responses resulting from exogenous substance P were abolished by atropine and TTX and enhanced by physostigmine. These findings suggest that the third phase may be mediated by the action of a substance P-like neuropeptide released from sensory nerve endings that subsequently activated cholinergic neurons. 2. The tachykinin receptors in the body of the guinea-pig oesophagus were characterized by determining the relative agonist potencies of natural tachykinins as well as tachykinin receptor-selective analogues. Antagonist affinities were also determined. The results indicated the presence of both NK2 and NK3 receptors. In addition, the effects of a cocktail of peptidase inhibitors (captopril, thiorphan and amastatin) on responses to various tachykinins and synthetic analogues were determined. The results indicate that one or more peptidases are present in this preparation. 3. Experiments using various tachykinin receptor antagonists were performed to determine whether the activation of tachykinin receptors played a role in the mediation of the third phase of the response to vagal nerve stimulation. While this response was unaffected by NK1 and NK2 receptor-selective antagonists, it was only partially inhibited (23%) by the NK3 receptor antagonist SR 142801. Thus, in the guinea-pig oesophagus, it appears that NK3 receptors play only a minor role in mediating a contractile response when afferent neurons are excited by vagal nerve stimulation.

Neurokinin-1 receptor antagonists for chemotherapy-induced nausea and vomiting.

PubMed

Aziz, Fahad

2012-07-01

Chemotherapy can be a life-prolonging treatment for many cancer patients, but it is often associated with profound nausea and vomiting that is so distressing that patients may delay or decline treatment to avoid these side effects. The discovery of several NK1 receptor antagonists is a big revolution to dealt this problem. NK1 receptor antagonists prevent both acute and delayed chemotherapy-induced nausea and vomiting (CINV). These agents act centrally at NK-1 receptors in vomiting centers within the central nervous system to block their activation by substance P released as an unwanted consequence of chemotherapy. By controlling nausea and vomiting, these agents help improve patients' daily living and their ability to complete multiple cycles of chemotherapy. They are effective for both moderately and highly emetogenic chemotherapy regimens. Their use might be associated with increased infection rates; however, additional appraisal of specific data from RCTs is needed.

A novel muscarinic receptor-independent mechanism of KCNQ2/3 potassium channel blockade by Oxotremorine-M.

PubMed

Zwart, Ruud; Reed, Hannah; Clarke, Sophie; Sher, Emanuele

2016-11-15

Inhibition of KCNQ (Kv7) potassium channels by activation of muscarinic acetylcholine receptors has been well established, and the ion currents through these channels have been long known as M-currents. We found that this cross-talk can be reconstituted in Xenopus oocytes by co-transfection of human recombinant muscarinic M1 receptors and KCNQ2/3 potassium channels. Application of the muscarinic acetylcholine receptor agonist Oxotremorine-methiodide (Oxo-M) between voltage pulses to activate KCNQ2/3 channels caused inhibition of the subsequent KCNQ2/3 responses. This effect of Oxo-M was blocked by the muscarinic acetylcholine receptor antagonist atropine. We also found that KCNQ2/3 currents were inhibited when Oxo-M was applied during an ongoing KCNQ2/3 response, an effect that was not blocked by atropine, suggesting that Oxo-M inhibits KCNQ2/3 channels directly. Indeed, also in oocytes that were transfected with only KCNQ2/3 channels, but not with muscarinic M1 receptors, Oxo-M inhibited the KCNQ2/3 response. These results show that besides the usual muscarinic acetylcholine receptor-mediated inhibition, Oxo-M also inhibits KCNQ2/3 channels by a direct mechanism. We subsequently tested xanomeline, which is a chemically distinct muscarinic acetylcholine receptor agonist, and oxotremorine, which is a close analogue of Oxo-M. Both compounds inhibited KCNQ2/3 currents via activation of M1 muscarinic acetylcholine receptors but, in contrast to Oxo-M, they did not directly inhibit KCNQ2/3 channels. Xanomeline and oxotremorine do not contain a positively charged trimethylammonium moiety that is present in Oxo-M, suggesting that such a charged moiety could be a crucial component mediating this newly described direct inhibition of KCNQ2/3 channels. Copyright © 2016 Elsevier B.V. All rights reserved.

Activation and desensitization of peripheral muscle and neuronal nicotinic acetylcholine receptors by selected, naturally-occurring pyridine alkaloids

USDA-ARS?s Scientific Manuscript database

Teratogenic alkaloids can cause developmental defects due to inhibition of fetal movement that results from desensitization of fetal muscletype nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiper...

Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

PubMed

Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

2017-06-05

Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Bone Morphogenetic Proteins, Antagonists and Receptors in Prostate Cancer

DTIC Science & Technology

2005-01-01

expressed in prostate. This work investigates BMP receptors and BMP antagonists to understand the basic mechanisms to inhibit the BMP signaling in...during embryoge- nesis, and prostate cancer metastases to bone. BMP functions can be inhibited by antagonists such as Noggin or DAN. DAN is a protein...protein along with a constant 0-6 -1 10 100 1000 1O0ng/ml of BMP-6, we were able to show a ng/ml BMP-6 dose-dependent inhibition of BMP-6 activity in DU

Mechanism of action of a nanomolar potent, allosteric antagonist of the thyroid-stimulating hormone receptor

PubMed Central

van Koppen, Chris J; de Gooyer, Marcel E; Karstens, Willem-Jan; Plate, Ralf; Conti, Paolo GM; van Achterberg, Tanja AE; van Amstel, Monique GA; Brands, Jolanda HGM; Wat, Jesse; Berg, Rob JW; Lane, J Robert D; Miltenburg, Andre MM; Timmers, C Marco

2012-01-01

BACKGROUND AND PURPOSE Graves' disease (GD) is an autoimmune disease in which the thyroid is overactive, producing excessive amounts of thyroid hormones, caused by thyroid-stimulating hormone (TSH) receptor-stimulating immunoglobulins (TSIs). Many GD patients also suffer from thyroid eye disease (Graves' ophthalmopathy or GO), as TSIs also activate TSH receptors in orbital tissue. We recently developed low molecular weight (LMW) TSH receptor antagonists as a novel therapeutic strategy for the treatment of GD and GO. Here, we determined the molecular pharmacology of a prototypic, nanomolar potent LMW TSH receptor antagonist, Org 274179-0. EXPERIMENTAL APPROACH Using CHO cells heterogeneously expressing human TSH receptors and rat FRTL-5 cells endogenously expressing rat TSH receptors, we determined the potency and efficacy of Org 274179-0 at antagonizing TSH- and TSI-induced TSH receptor signalling and its cross-reactivity at related follicle-stimulating hormone and luteinizing hormone receptors. We analysed the allosteric mode of interaction of Org 274179-0 and determined whether it is an inverse agonist at five naturally occurring, constitutively active TSH receptor mutants. KEY RESULTS Nanomolar concentrations of Org 274179-0 completely inhibited TSH (and TSI)-mediated TSH receptor activation with little effect on the potency of TSH, in accordance with an allosteric mechanism of action. Conversely, increasing levels of TSH receptor stimulation only marginally reduced the antagonist potency of Org 274179-0. Org 274179-0 fully blocked the increased basal activity of all the constitutively active TSH receptor mutants tested with nanomolar potencies. CONCLUSIONS AND IMPLICATIONS Nanomolar potent TSH receptor antagonists like Org 274179-0 have therapeutic potential for the treatment of GD and GO. PMID:22014107

Tryptophanol-derived oxazolopiperidone lactams: identification of a hit compound as NMDA receptor antagonist.

PubMed

Pereira, Nuno A L; Sureda, Francesc X; Esplugas, Roser; Pérez, Maria; Amat, Mercedes; Santos, Maria M M

2014-08-01

N-Methyl-D-aspartate receptors (NMDAR) exacerbated activation leads to neuron death through a phenomenon called excitotoxicity. These receptors are implicated in several neurological diseases (e.g., Alzheimer and Parkinson) and thus represent an important therapeutic target. We herein describe the study of enantiopure tryptophanol-derived oxazolopiperidone lactams as NMDA receptor antagonists. The most active hit exhibited an IC50 of 63.4 μM in cultured rat cerebellar granule neurons thus being 1.5 fold more active than clinically approved NMDA antagonist amantadine (IC50=92 μM). Copyright © 2014 Elsevier Ltd. All rights reserved.

The 3,7-diazabicyclo[3.3.1]nonane scaffold for subtype selective nicotinic acetylcholine receptor ligands. Part 2: carboxamide derivatives with different spacer motifs.

PubMed

Eibl, Christoph; Munoz, Lenka; Tomassoli, Isabelle; Stokes, Clare; Papke, Roger L; Gündisch, Daniela

2013-12-01

3,7-Diazabicyclo[3.3.1]nonane (bispidine) based nicotinic acetylcholine receptor (nAChR) ligands have been synthesized and evaluated for nAChRs interaction. Diverse spacer motifs were incorporated between the hydrogen bond acceptor (HBA) part and a variety of substituted (hetero)aryl moieties. Bispidine carboxamides bearing spacer motifs often showed high affinity in the low nanomolar range and selectivity for the α4β2(∗) nAChR. Compounds 15, 25, and 47 with Ki values of about 1 nM displayed the highest affinities for α4β2(∗) nAChR. All evaluated compounds are partial agonists or antagonists at α4β2(∗), with reduced or no effects on α3β4(∗) with the exception of compound 15 (agonist), and reduced or no effect at α7 and muscle subtypes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis and pharmacology of alkanediguanidinium compounds that block the neuronal nicotinic acetylcholine receptor.

PubMed

Villarroya, M; Gandía, L; López, M G; García, A G; Cueto, S; García-Navio, J L; Alvarez-Builla, J

1996-08-01

Taking as models the polyamine toxin fraction FTX from the funnel-web spider venom, and the guanidinium moiety of guanethidine, a series of azaalkane-1, omega-diguanidinium salts were obtained. Some of them blocked ion fluxes through the neuronal nicotinic receptors for acetylcholine (nAChR). The blockade was exerted at submicromolar concentrations, suggesting a highly selective interaction with the nAChR. In fact, the active compounds on the nAChR ion channel did not recognize the voltage-dependent Na+ or Ca2+ channels of bovine adrenal chromaffin cells. Therefore, these compounds may be useful tools to clarify the functions of nAChR receptors in the central and peripheral nervous systems.

5-HT2A receptor antagonists improve motor impairments in the MPTP mouse model of Parkinson's disease.

PubMed

Ferguson, Marcus C; Nayyar, Tultul; Deutch, Ariel Y; Ansah, Twum A

2010-01-01

Clinical observations have suggested that ritanserin, a 5-HT(2A/C) receptor antagonist may reduce motor deficits in persons with Parkinson's Disease (PD). To better understand the potential antiparkinsonian actions of ritanserin, we compared the effects of ritanserin with the selective 5-HT(2A) receptor antagonist M100907 and the selective 5-HT(2C) receptor antagonist SB 206553 on motor impairments in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-treated mice exhibited decreased performance on the beam-walking apparatus. These motor deficits were reversed by acute treatment with L-3,4-dihydroxyphenylalanine (levodopa). Both the mixed 5-HT(2A/C) antagonist ritanserin and the selective 5-HT(2A) antagonist M100907 improved motor performance on the beam-walking apparatus. In contrast, SB 206553 was ineffective in improving the motor deficits in MPTP-treated mice. These data suggest that 5-HT(2A) receptor antagonists may represent a novel approach to ameliorate motor symptoms of Parkinson's disease. Published by Elsevier Ltd.

5-HT2A receptor antagonists improve motor impairments in the MPTP mouse model of Parkinson's disease

PubMed Central

Ferguson, Marcus C.; Nayyar, Tultul; Deutch, Ariel Y.; Ansah, Twum A.

2010-01-01

Clinical observations have suggested that ritanserin, a 5-HT2A/C receptor antagonist may reduce motor deficits in persons with Parkinson's Disease (PD). To better understand the potential antiparkinsonian actions of ritanserin, we compared the effects of ritanserin with the selective 5-HT2A receptor antagonist M100907 and the selective 5-HT2C receptor antagonist SB 206553 on motor impairments in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-treated mice exhibited decreased performance on the beam-walking apparatus. These motor deficits were reversed by acute treatment with L-3,4-dihydroxyphenylalanine (levodopa). Both the mixed 5-HT2A/C antagonist ritanserin and the selective 5-HT2A antagonist M100907 improved motor performance on the beam-walking apparatus. In contrast, SB 206553 was ineffective in improving the motor deficits in MPTP-treated mice. These data suggest that 5-HT2A receptor antagonists may represent a novel approach to ameliorate motor symptoms of Parkinson's disease. PMID:20361986

AM-37 and ST-36 Are Small Molecule Bombesin Receptor Antagonists

PubMed Central

Moody, Terry W.; Tashakkori, Nicole; Mantey, Samuel A.; Moreno, Paola; Ramos-Alvarez, Irene; Leopoldo, Marcello; Jensen, Robert T.

2017-01-01

While peptide antagonists for the gastrin-releasing peptide receptor (BB2R), neuromedin B receptor (BB1R), and bombesin (BB) receptor subtype-3 (BRS-3) exist, there is a need to develop non-peptide small molecule inhibitors for all three BBR. The BB agonist (BA)1 binds with high affinity to the BB1R, BB2R, and BRS-3. In this communication, small molecule BBR antagonists were evaluated using human lung cancer cells. AM-37 and ST-36 inhibited binding to human BB1R, BB2R, and BRS-3 with similar affinity (Ki = 1.4–10.8 µM). AM-13 and AM-14 were approximately an order of magnitude less potent than AM-37 and ST-36. The ability of BA1 to elevate cytosolic Ca2+ in human lung cancer cells transfected with BB1R, BB2R, and BRS-3 was antagonized by AM-37 and ST-36. BA1 increased tyrosine phosphorylation of the EGFR and ERK in lung cancer cells, which was blocked by AM-37 and ST-36. AM-37 and ST-36 reduced the growth of lung cancer cells that have BBR. The results indicate that AM-37 and ST-36 function as small molecule BB receptor antagonists. PMID:28785244

Structural characterization of agonist and antagonist-bound acetylcholine-binding protein from Aplysia californica.

PubMed

Hansen, Scott B; Sulzenbacher, Gerlind; Huxford, Tom; Marchot, Pascale; Bourne, Yves; Taylor, Palmer

2006-01-01

Nicotinic acetylcholine receptors (nAChRs) are well-characterized allosteric transmembrane proteins involved in the rapid gating of ions elicited by ACh. These receptors belong to the Cys-loop superfamily of ligand-gated ion channels, which also includes GABAA and GABAC, 5-HT3, and glycine receptors. The nAChRs are homo- or heteromeric pentamers of structurally related subunits that encompass an extracellular N-terminal ligand-binding domain, four transmembrane-spanning regions that form the ion channel, and an extended intracellular region between spans 3 and 4. Ligand binding triggers conformational changes that are transmitted to the transmembrane-spanning region, leading to gating and changes in membrane potential. The four transmembrane spans on each of the five subunits create a substantial region of hydrophobicity that precludes facile crystallization of this protein. However the freshwater snail, Lymnaea stagnalis, produces a soluble homopentameric protein, termed the ACh-binding protein (AChBP), which binds ACh (Smit et al., 2001). Its structure was determined recently (Brejc et al., 2001) at high resolution, revealing the structural scaffold for nAChR, and has become a functional and structural surrogate of the nAChR ligand-binding domain. We have characterized an AChBP from Aplysia californica and determined distinct ligand-binding properties when compared to those of L. stagnalis, including ligand specificity for the nAChR alpha7 subtype-specific alpha-conotoxin ImI (Hansen et al., 2004).

The utility of ionotropic glutamate receptor antagonists in the treatment of nociception induced by epidural glutamate infusion in rats.

PubMed

Osgood, Doreen B; Harrington, William F; Kenney, Elizabeth V; Harrington, J Frederick

2013-01-01

The authors have previously demonstrated that human herniated disc material contains high concentrations of free glutamate. In an experimental model, elevated epidural glutamate concentrations in the lumbar spine can cause a focal hyperesthetic state. Rats underwent epidural glutamate infusion in the lumbar spine by a miniosmotic pump over a 72-hour period. Some rats underwent coinfusion with glutamate and ionotropic glutamate antagonists. Nociception was assessed by von Frey fibers and by assessment of glutamate receptor expression in the corresponding dorsal horn of the spinal cord. The kainic acid antagonist, UBP 301, decreased epidural glutamate-based hyperesthesia in a dose dependent manner. Concordant with these findings, there was significant decrease in kainate receptor expression in the dorsal horn. The N-Methyl-4-isoxazoleproionic acid (NMDA) antagonist Norketamine also significantly diminished hyperesthesia and decreased receptor expression in the dorsal horn. Both UBP 301, the kainic acid receptor antagonist and Norketamine, an NMDA receptor antagonist, dampened epidural glutamate-based nociception. Focal epidural injections of Kainate or NMDA receptor antagonists could be effective treatments for disc herniation-based lumbar radiculopathy.

The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype.

PubMed

Killen, J A; Hochschwender, S M; Lindstrom, J M

1985-08-01

Anti-idiotype antibodies were induced in rats by immunization with rat monoclonal antibodies to the main immunogenic region of acetylcholine receptors. These anti-idiotype antibodies showed very little crossreaction with other rat monoclonal antibodies which bind to the same region of the receptor. When the rats producing these anti-idiotype antibodies were immunized with receptor, they showed no net decrease in anti-receptor antibody production. These data indicate that, although more than half of the antibodies produced by rats immunized with receptor are directed at a small region, many anti-receptor idiotypes are involved in this response and anti-idiotype therapy is not beneficial.

Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bencherif, M.; Lukas, R.J.

1991-06-01

Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10more » microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.« less

IPPA08 allosterically enhances the action of imidacloprid on nicotinic acetylcholine receptors.

PubMed

Bao, Haibo; Shao, Xusheng; Zhang, Yixi; Cheng, Jiagao; Wang, Yunchao; Xu, Xiaoyong; Fang, Jichao; Liu, Zewen; Li, Zhong

2016-12-01

Our previous study showed that IPPA08, a cis-configuration neonicotinoid compound with unique oxabridged substructure, acted as a specific synergist to neonicotinoid insecticides targeting nicotinic acetylcholine receptors (nAChRs). Heteropentamer nAChRs have diverse characteristics and can form canonical and noncanonical subunit interfaces. While canonical interfaces have been exploited as targets of many drugs, noncanonical interfaces have received less attention. In this study, the mechanism of IPPA08 synergism was evaluated on hybrid nAChRs consisting of three α1 subunits from the brown planthopper and two rat β1 subunits (Nlα1/rβ2) expressed in Xenopus oocytes. IPPA08 alone evoked inward currents, but only at very high concentrations, greater than 1 mM. However, at concentrations below 200 μM, IPPA08 slowed the decay of inward currents evoked by imidacloprid, but not by acetylcholine, and also increased the sensitivity of Nlα1/rβ2 to imidacloprid. Both modulations by IPPA08 were concentration-dependent in the same concentration range of 10-150 μM. Experimentally induced mutations in canonical (α+/β-) and noncanonical (β+/α-) interfaces of Nlα1/rβ2 receptors were also examined to evaluate the presence of possible binding sites for IPPA08 on the receptors. Our results showed that mutations in the canonical interfaces affected only the potency of IPPA08 as an agonist, while mutations in the noncanonical interfaces affected only the synergistic action of IPPA08. Based on these results, we propose that at low concentrations IPPA08 can act as a positive allosteric modulator of noncanonical interfaces, and likely slow the decay of currents through stabilizing the open-channel state caused by the action of imidacloprid on canonical interfaces. Copyright © 2016 Elsevier Ltd. All rights reserved.

Potential antipsychotic properties of central cannabinoid (CB1) receptor antagonists.

PubMed

Roser, Patrik; Vollenweider, Franz X; Kawohl, Wolfram

2010-03-01

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC), the principal psychoactive constituent of the Cannabis sativa plant, and other agonists at the central cannabinoid (CB(1)) receptor may induce characteristic psychomotor effects, psychotic reactions and cognitive impairment resembling schizophrenia. These effects of Delta(9)-THC can be reduced in animal and human models of psychopathology by two exogenous cannabinoids, cannabidiol (CBD) and SR141716. CBD is the second most abundant constituent of Cannabis sativa that has weak partial antagonistic properties at the CB(1) receptor. CBD inhibits the reuptake and hydrolysis of anandamide, the most important endogenous CB(1) receptor agonist, and exhibits neuroprotective antioxidant activity. SR141716 is a potent and selective CB(1) receptor antagonist. Since both CBD and SR141716 can reverse many of the biochemical, physiological and behavioural effects of CB(1) receptor agonists, it has been proposed that both CBD and SR141716 have antipsychotic properties. Various experimental studies in animals, healthy human volunteers, and schizophrenic patients support this notion. Moreover, recent studies suggest that cannabinoids such as CBD and SR141716 have a pharmacological profile similar to that of atypical antipsychotic drugs. In this review, both preclinical and clinical studies investigating the potential antipsychotic effects of both CBD and SR141716 are presented together with the possible underlying mechanisms of action.

Tropisetron sensitizes α7 containing nicotinic receptors to low levels of acetylcholine in vitro and improves memory-related task performance in young and aged animals.

PubMed

Callahan, Patrick M; Bertrand, Daniel; Bertrand, Sonia; Plagenhoef, Marc R; Terry, Alvin V

2017-05-01

Tropisetron, a 5-HT 3 receptor antagonist commonly prescribed for chemotherapy-induced nausea and vomiting also exhibits high affinity, partial agonist activity at α7 nicotinic acetylcholine receptors (α7 nAChRs). α7 nAChRs are considered viable therapeutic targets for neuropsychiatric disorders such as Alzheimer's disease (AD). Here we further explored the nAChR pharmacology of tropisetron to include the homomeric α7 nAChR and recently characterized heteromeric α7β2 nAChR (1:10 ratio) and we evaluated its cognitive effects in young and aged animals. Electrophysiological studies on human nAChRs expressed in Xenopus oocytes confirmed the partial agonist activity of tropisetron at α7 nAChRs (EC 50 ∼2.4 μM) with a similar effect at α7β2 nAChRs (EC 50 ∼1.5 μM). Moreover, currents evoked by irregular pulses of acetylcholine (40 μM) at α7 and α7β2 nAChRs were enhanced during sustained exposure to low concentrations of tropisetron (10 and 30 nM) indicative of a "priming" or co-agonist effect. Tropisetron (0.1-10 mg/kg) improved novel object recognition performance in young Sprague-Dawley rats and in aged Fischer rats. In aged male and female rhesus monkeys, tropisetron (0.03-1 mg/kg) produced a 17% increase from baseline levels in delayed match to sample long delay accuracy while combination of non-effective doses of donepezil (0.1 mg/kg) and tropisetron (0.03 and 0.1 mg/kg) produced a 24% change in accuracy. Collectively, these animal experiments indicate that tropisetron enhances cognition and has the ability to improve the effective dose range of currently prescribed AD therapy (donepezil). Moreover, these effects may be explained by tropisetron's ability to sensitize α7 containing nAChRs to low levels of acetylcholine. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

PubMed

Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

2017-01-01

Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates.

PubMed

Shen, Xin-Ming; Brengman, Joan; Neubauer, David; Sine, Steven M; Engel, Andrew G

2016-02-12

We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Age-related changes in functional postsynaptic nicotinic acetylcholine receptor subunits in neurons of the laterodorsal tegmental nucleus, a nucleus important in drug addiction.

PubMed

Christensen, Mark H; Kohlmeier, Kristi A

2016-03-01

The earlier an individual initiates cigarette smoking, the higher the likelihood of development of dependency to nicotine, the addictive ingredient in cigarettes. One possible mechanism underlying this higher addiction liability is an ontogenetically differential cellular response induced by nicotine in neurons mediating the reinforcing or euphoric effects of this drug, which could arise from age-related differences in the composition of nicotinic acetylcholine receptor (nAChR) subunits. In the current study, we examined whether the subunit composition of nAChRs differed between neurons within the laterodorsal tegmentum (LDT), a nucleus importantly involved in drug addiction associated behaviours, across two periods of ontogeny in which nicotine-mediated excitatory responses were shown to depend on age. To this end, whole-cell patch-clamp recordings in mouse brain slices from identified LDT neurons, in combination with nAChR subunit-specific receptor antagonists, were conducted. Comparison of the contribution of different nAChR subunits to acetylcholine (ACh)-induced inward currents indicated that the contributions of the β2 and/or β4 and α7 nAChR subunits alter across age. Taken together, we conclude that across a limited ontogenetic period, there is plasticity in the subunit composition of nAChRs in LDT neurons. In addition, our data indicate, for the first time, functional presence of α6 nAChR subunits in LDT neurons within the age ranges studied. Changes in subunit composition of nAChRs across ontogeny could contribute to the age-related differential excitability induced by nicotine. Differences in the subunit composition of nAChRs within the LDT would be expected to contribute to ontogenetic-dependent outflow from the LDT to target regions, which include reward-related circuitry. © 2014 Society for the Study of Addiction.

Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors.

PubMed

Disingrini, Teresa; Muth, Mathias; Dallanoce, Clelia; Barocelli, Elisabetta; Bertoni, Simona; Kellershohn, Kerstin; Mohr, Klaus; De Amici, Marco; Holzgrabe, Ulrike

2006-01-12

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

Enhanced Chronic Pain Management Utilizing Chemokine Receptor Antagonists

DTIC Science & Technology

2016-08-01

approximately halfway into the solution. All animals were tested at 60, 15 and 0 min before drug injection. For each animal , the first reading was discarded...approval (December 31, 2015), hiring new personnel, conducting baseline testing for procedures not involving animals , testing equipment, developing...treatment; Analgesia; Nociception; Antinociception; Inflammation; Chemokines; Chemokine receptor antagonists; Opioid analgesics; Animal models of pain

A novel human-based receptor antagonist of sustained action reveals body weight control by endogenous GLP-1.

PubMed

Patterson, James T; Ottaway, Nickki; Gelfanov, Vasily M; Smiley, David L; Perez-Tilve, Diego; Pfluger, Paul T; Tschöp, Matthias H; Dimarchi, Richard D

2011-02-18

Ex-4 (9-39)a is a well characterized GLP-1 receptor antagonist that suffers from two notable limitations, its nonhuman amino acid sequence and its relatively short in vivo duration of action. Comparable N-terminal shortening of human GLP-1 lessens agonism but does not provide a high potency antagonist. Through a series of GLP-1/Ex-4 hybrid peptides, the minimal structural changes required to generate a pure GLP-1-based antagonist were identified as Glu16, Val19, and Arg20, yielding an antagonist of approximately 3-fold greater in vitro potency compared with Ex-4 (9-39)a. The structural basis of antagonism appears to result from stabilization of the α helix combined with enhanced electrostatic and hydrophobic interactions with the extracellular domain of the receptor. Site-specific acylation of the human-based antagonist yielded a peptide of increased potency as a GLP-1 receptor antagonist and 10-fold greater selectivity relative to the GIP receptor. The acylated antagonist demonstrated sufficient duration of action to maintain inhibitory activity when administered as a daily subcutaneous injection. The sustained pharmacokinetics and enhanced human sequence combine to form an antagonist optimized for clinical study. Daily administration of this antagonist by subcutaneous injection to diet-induced obese mice for 1 week caused a significant increase in food intake, body weight, and glucose intolerance, demonstrating endogenous GLP-1 as a relevant hormone in mammalian energy balance in the obese state.

A review of granisetron, 5-hydroxytryptamine3 receptor antagonists, and other antiemetics.

PubMed

Hsu, Eric S

2010-01-01

Nausea and vomiting are 2 of the most upsetting adverse reactions of chemotherapy. Current guidelines propose 5-hydroxytryptamine3 (5-HT3) receptor antagonists as a pharmacologic intervention for acute and delayed nausea and vomiting [chemotherapy-induced nausea and vomiting (CINV)] associated with moderately and highly emetogenic chemotherapy. Meanwhile, both postoperative nausea and vomiting (PONV) and postdischarge nausea and vomiting are challenging situations after surgeries and procedures. Prophylactic and therapeutic combinations of antiemetics are recommended in patients at high risk of suffering from PONV and postdischarge nausea and vomiting. Granisetron (Kytril) is a selective 5-HT3 receptor antagonist that does not induce or inhibit the hepatic cytochrome P-450 system in vitro. There are also 4 other antagonists of 5-HT3 receptor (dolasetron, ondansetron, palonosetron, and tropisetron) being metabolized via the CYP2D6 and are subject to potential genetic polymorphism. The launch of a new class of antiemetics, the substance P/neurokinin1 receptor antagonists, was attributed to the scientific update on the central generator responsible for emesis and role of substance P. There has been mounting interest in exploring integrative medicine, either acupuncture or acustimulation of P6 (Nei-Kuwan), to complement the western medicine for prevention and management of nausea and vomiting. The potential application of cannabinoids, either alone or in combination with other agents of different mechanism, could contribute further to improve outcome in CINV. Implementation of future treatment guidelines for more effective management of CINV and PONV could certainly improve the efficacy and outcome of cancer and postoperative care.

Common influences of non-competitive NMDA receptor antagonists on the consolidation and reconsolidation of cocaine-cue memory.

PubMed

Alaghband, Yasaman; Marshall, John F

2013-04-01

Environmental stimuli or contexts previously associated with rewarding drugs contribute importantly to relapse among addicts, and research has focused on neurobiological processes maintaining those memories. Much research shows contributions of cell surface receptors and intracellular signaling pathways in maintaining associations between rewarding drugs (e.g., cocaine) and concurrent cues/contexts; these memories can be degraded at the time of their retrieval through reconsolidation interference. Much less studied is the consolidation of drug-cue memories during their acquisition. The present experiments use the cocaine-conditioned place preference (CPP) paradigm in rats to directly compare, in a consistent setting, the effects of N-methyl-D-aspartate (NMDA) glutamate receptor antagonists MK-801 and memantine on the consolidation and reconsolidation of cocaine-cue memories. For the consolidation studies, animals were systemically administered MK-801 or memantine immediately following training sessions. To investigate the effects of these NMDA receptor antagonists on the retention of previously established cocaine-cue memories, animals were systemically administered MK-801 or memantine immediately after memory retrieval. Animals given either NMDA receptor antagonist immediately following training sessions did not establish a preference for the cocaine-paired compartment. Post-retrieval administration of either NMDA receptor antagonist attenuated the animals' preference for the cocaine-paired compartment. Furthermore, animals given NMDA receptor antagonists post-retrieval showed a blunted response to cocaine-primed reinstatement. Using two distinct NMDA receptor antagonists in a common setting, these findings demonstrate that NMDA receptor-dependent processes contribute both to the consolidation and reconsolidation of cocaine-cue memories, and they point to the potential utility of treatments that interfere with drug-cue memory reconsolidation.

Acetylcholine-induced seizure-like activity and modified cholinergic gene expression in chronically epileptic rats.

PubMed

Zimmerman, Gabriel; Njunting, Marleisje; Ivens, Sebastian; Tolner, Else A; Tolner, Elsa; Behrens, Christoph J; Gross, Miriam; Soreq, Hermona; Heinemann, Uwe; Friedman, Alon

2008-02-01

The entorhinal cortex (EC) plays an important role in temporal lobe epilepsy. Under normal conditions, the enriched cholinergic innervation of the EC modulates local synchronized oscillatory activity; however, its role in epilepsy is unknown. Enhanced neuronal activation has been shown to induce transcriptional changes of key cholinergic genes and thus alter cholinergic responses. To examine cholinergic modulations in epileptic tissue we studied molecular and electrophysiological cholinergic responses in the EC of chronically epileptic rats following exposure to pilocarpine or kainic acid. We confirmed that while the total activity of the acetylcholine (ACh)-hydrolysing enzyme, acetylcholinesterase (AChE) was not altered, epileptic rats showed alternative splicing of AChE pre-mRNA transcripts, accompanied by a shift from membrane-bound AChE tetramers to soluble monomers. This was associated with increased sensitivity to ACh application: thus, in control rats, ACh (10-100 microm) induced slow (< 1Hz), periodic events confined to the EC; however, in epileptic rats, ACh evoked seconds-long seizure-like events with initial appearance in the EC, and frequent propagation to neighbouring cortical regions. ACh-induced seizure-like events could be completely blocked by the non-specific muscarinic antagonist, atropine, and were partially blocked by the muscarinic-1 receptor antagonist, pirenzepine; but were not affected by the non-specific nicotinic antagonist, mecamylamine. Epileptic rats presented reduced transcript levels of muscarinic receptors with no evidence of mRNA editing or altered mRNA levels for nicotinic ACh receptors. Our findings suggest that altered cholinergic modulation may initiate seizure events in the epileptic temporal cortex.

Combination decongestion therapy in hospitalized heart failure: loop diuretics, mineralocorticoid receptor antagonists and vasopressin antagonists.

PubMed

Vaduganathan, Muthiah; Mentz, Robert J; Greene, Stephen J; Senni, Michele; Sato, Naoki; Nodari, Savina; Butler, Javed; Gheorghiade, Mihai

2015-01-01

Congestion is the most common reason for admissions and readmissions for heart failure (HF). The vast majority of hospitalized HF patients appear to respond readily to loop diuretics, but available data suggest that a significant proportion are being discharged with persistent evidence of congestion. Although novel therapies targeting congestion should continue to be developed, currently available agents may be utilized more optimally to facilitate complete decongestion. The combination of loop diuretics, natriuretic doses of mineralocorticoid receptor antagonists and vasopressin antagonists represents a regimen of currently available therapies that affects early and persistent decongestion, while limiting the associated risks of electrolyte disturbances, hemodynamic fluctuations, renal dysfunction and mortality.

The mGlu5 receptor antagonist MPEP activates specific stress-related brain regions and lacks neurotoxic effects of the NMDA receptor antagonist MK-801: significance for the use as anxiolytic/antidepressant drug.

PubMed

Inta, Dragos; Filipovic, Dragana; Lima-Ojeda, Juan M; Dormann, Christof; Pfeiffer, Natascha; Gasparini, Fabrizio; Gass, Peter

2012-04-01

Glutamatergic agents have been conceptualized as powerful, fast-acting alternatives to monoaminergic-based antidepressants. NMDA receptor antagonists such as ketamine or MK-801 are therapeutically effective, but their clinical use is hampered by psychotomimetic effects, accompanied by neurotoxicity in the retrosplenial and cingulate cortex. Antagonists of metabotropic mGlu5 receptors like MPEP elicit both robust antidepressant and anxiolytic effects; however, the underlying mechanisms are yet unknown. mGlu5 receptors closely interact with NMDA receptors, but whether MPEP induces neurotoxicity similar to NMDA receptor antagonists has not been elucidated. We show here using c-Fos brain mapping that MPEP administration results in a restricted activation of distinct stress-related brain areas, including the bed nucleus of stria terminalis (BNST), central nucleus of the amygdala, and paraventricular nucleus of the hypothalamus (PVNH), in a pattern similar to that induced by classical antidepressants and anxiolytics. Unlike the NMDA antagonist MK-801, MPEP does not injure the adult retrosplenial cortex, in which it fails to induce heat shock protein 70 (Hsp70). Moreover, MPEP does not elicit to the same extent as MK-801 apoptosis in cortical areas at perinatal stages, as revealed by caspase 3 expression. These data identify new cellular targets for the anxiolytic and antidepressant effect of MPEP, indicating also in addition that in contrast to MK-801, it lacks the cortical neurotoxicity associated with psychotomimetic side-effects. Copyright © 2012 Elsevier Ltd. All rights reserved.

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

PubMed Central

Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

2015-01-01

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

Caffeine and Selective Adenosine Receptor Antagonists as New Therapeutic Tools for the Motivational Symptoms of Depression

PubMed Central

López-Cruz, Laura; Salamone, John D.; Correa, Mercè

2018-01-01

Major depressive disorder is one of the most common and debilitating psychiatric disorders. Some of the motivational symptoms of depression, such anergia (lack of self-reported energy) and fatigue are relatively resistant to traditional treatments such as serotonin uptake inhibitors. Thus, new pharmacological targets are being investigated. Epidemiological data suggest that caffeine consumption can have an impact on aspects of depressive symptomatology. Caffeine is a non-selective adenosine antagonist for A1/A2A receptors, and has been demonstrated to modulate behavior in classical animal models of depression. Moreover, selective adenosine receptor antagonists are being assessed for their antidepressant effects in animal studies. This review focuses on how caffeine and selective adenosine antagonists can improve different aspects of depression in humans, as well as in animal models. The effects on motivational symptoms of depression such as anergia, fatigue, and psychomotor slowing receive particular attention. Thus, the ability of adenosine receptor antagonists to reverse the anergia induced by dopamine antagonism or depletion is of special interest. In conclusion, although further studies are needed, it appears that caffeine and selective adenosine receptor antagonists could be therapeutic agents for the treatment of motivational dysfunction in depression. PMID:29910727

Modal gating of muscle nicotinic acetylcholine receptors

NASA Astrophysics Data System (ADS)

Vij, Ridhima

Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

Effect of glutamate receptor antagonists and antirheumatic drugs on proliferation of synoviocytes in vitro.

PubMed

Parada-Turska, Jolanta; Rzeski, Wojciech; Majdan, Maria; Kandefer-Szerszeń, Martyna; Turski, Waldemar A

2006-03-27

One of the most striking features of inflammatory arthritis is the hyperplasia of synovial fibroblasts. It is not known whether the massive synovial hyperplasia characteristic of rheumatoid arthritis is due to the proliferation of synovial fibroblasts or to defective apoptosis. It has been found that glutamate receptor antagonists inhibit proliferation of different human tumour cells and the anticancer potential of glutamate receptor antagonists was suggested. Here, we investigated the effect of glutamate receptor antagonists and selected antirheumatic drugs on proliferation of synoviocytes in vitro. Experiments were conducted on rabbit synoviocytes cell line HIG-82 obtained from American Type Culture Collection (Menassas, VA, USA). Cell proliferation was assessed by means of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 value (the concentration of drug necessary to induce 50% inhibition) together with confidence limits was calculated. Glutamate receptor antagonists, 1-(4-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-one (CFM-2), riluzole, memantine, 1-4-aminophenyl-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), dizocilpine, ketamine and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), inhibited proliferation of synoviocytes with the following IC50 values (in mM): 0.014, 0.017, 0.065, 0.102, 0.15, 0.435 and 1.16, respectively. Antirheumatic drugs, celecoxib, diclofenac, nimesulide, sulfasalazine, naproxen and methotrexate, inhibited proliferation of synoviocytes with the following IC50 values (in mM): 0.0043, 0.034, 0.044, 0.096, 0.385 and 1.123, respectively. Thus, the antiproliferative potential of glutamate receptor antagonists is comparable to that of antirheumatic drugs.

In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

NASA Astrophysics Data System (ADS)

Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

1983-05-01

A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

Characterization of the tachykinin neurokinin-2 receptor in the human urinary bladder by means of selective receptor antagonists and peptidase inhibitors.

PubMed

Giuliani, S; Patacchini, R; Barbanti, G; Turini, D; Rovero, P; Quartara, L; Giachetti, A; Maggi, C A

1993-11-01

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

PubMed Central

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jurgen; Kobilka, Brian

2012-01-01

Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors. PMID:22358844

Nonsteroidal antagonists of the mineralocorticoid receptor.

PubMed

Kolkhof, Peter; Nowack, Christina; Eitner, Frank

2015-09-01

The broad clinical use of steroidal mineralocorticoid receptor antagonists (MRAs) is limited by the potential risk of inducing hyperkalemia when given on top of renin-angiotensin system blockade. Drug discovery campaigns have been launched aiming for the identification of nonsteroidal MRAs with an improved safety profile. This review analyses the evidence for the potential of improved safety profiles of nonsteroidal MRAs and the current landscape of clinical trials with nonsteroidal MRAs. At least three novel nonsteroidal MRAs have reportedly demonstrated an improved therapeutic index (i.e. less risk for hyperkalemia) in comparison to steroidal antagonists in preclinical models. Five pharmaceutical companies have nonsteroidal MRAs in clinical development with a clear focus on the treatment of chronic kidney diseases. No clinical data have been published so far for MT-3995 (Mitsubishi), SC-3150 (Daiichi-Sankyo), LY2623091 (Eli Lilly) and PF-03882845 (Pfizer). In contrast, data from two clinical phase II trials are available for finerenone (Bayer) which demonstrated safety and efficacy in patients with heart failure and additional chronic kidney diseases, and significantly reduced albuminuria in patients with diabetic nephropathy. Neither hyperkalemia nor reductions in kidney function were limiting factors to its use. Novel, nonsteroidal MRAs are currently tested in clinical trials. Based on preclinical and first clinical data, these nonsteroidal MRAs might overcome the limitations of today's steroidal antagonists.

Phosphocholine - an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors.

PubMed

Richter, K; Mathes, V; Fronius, M; Althaus, M; Hecker, A; Krasteva-Christ, G; Padberg, W; Hone, A J; McIntosh, J M; Zakrzewicz, A; Grau, V

2016-06-28

We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions.

Sigma1 receptor antagonists determine the behavioral pattern of the methamphetamine-induced stereotypy in mice

PubMed Central

Kitanaka, J.; Kitanaka, N.; Tatsuta, T.; Hall, F.S.; Uhl, G.R.; Tanaka, K.; Nishiyama, N.; Morita, Y.; Takemura, M.

2011-01-01

Objective The effects of sigma receptor antagonists on methamphetamine (METH)-induced stereotypy have not been examined. We examined the effects of sigma antagonists on METH-induced stereotypy in mice. Results The administration of METH (10 mg/kg) to male ddY mice induced stereotyped behavior consisting of biting (90.1%), sniffing (4.2%), head bobbing (4.1%), and circling (1.7%) during an observation period of 1 h. Pretreatment of the mice with BMY 14802 (α-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazinebutanol; 1, 5, and 10 mg/kg), a non-specific sigma receptor antagonist, significantly increased METH-induced sniffing (19.2, 30.5, and 43.8% of total stereotypical behavior) but decreased biting (76.6, 66.9, and 49.3% of total stereotypical behavior) in a dose-dependent manner. This response was completely abolished by (+)-SKF 10,047 ([2S-(2α,6α,11R)]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol; 4 and 10 mg/kg), a putative sigma1 receptor agonist, and partially by PB 28 (1-cyclohexyl-4-[3-(1,2,3,4-tetrahydro-5-methoxy-1-naphthalen-1-yl)-n-propyl]piperazine; 1 and 10 mg/kg), a putative sigma2 receptor agonist. The BMY 14802 action on METH-induced stereotypy was mimicked by BD 1047 (N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine; 10 mg/kg), a putative sigma1 receptor antagonist, but not by SM-21 ((±)-tropanyl 2-(4-chlorophenoxy)butanoate; 1 mg/kg), a putative sigma2 receptor antagonist. The BD 1047 effect on METH-induced stereotypy was also abolished completely by (+)-SKF 10,047 and partially by PB 28. The overall frequency of METH-induced stereotypical behavior was unchanged with these sigma receptor ligands, despite the alteration in particular behavioral patterns. The BMY 14802 action on METH-induced stereotypy was unaffected by pretreatment with centrally acting histamine H1 receptor antagonists (pyrilamine or ketotifen, 10 mg/kg), suggesting that these effects are independent of histamine H1

Cost-effectiveness of histamine receptor-2 antagonist versus proton pump inhibitor for stress ulcer prophylaxis in critically ill patients*.

PubMed

MacLaren, Robert; Campbell, Jon

2014-04-01

To examine the cost-effectiveness of using histamine receptor-2 antagonist or proton pump inhibitor for stress ulcer prophylaxis. Decision analysis model examining costs and effectiveness of using histamine receptor-2 antagonist or proton pump inhibitor for stress ulcer prophylaxis. Costs were expressed in 2012 U.S. dollars from the perspective of the institution and included drug regimens and the following outcomes: clinically significant stress-related mucosal bleed, ventilator-associated pneumonia, and Clostridium difficile infection. Effectiveness was the mortality risk associated with these outcomes and represented by survival. Costs, occurrence rates, and mortality probabilities were extracted from published data. A simulation model. A mixed adult ICU population. Histamine receptor-2 antagonist or proton pump inhibitor for 9 days of stress ulcer prophylaxis therapy. Output variables were expected costs, expected survival rates, incremental cost, and incremental survival rate. Univariate sensitivity analyses were conducted to determine the drivers of incremental cost and incremental survival. Probabilistic sensitivity analysis was conducted using second-order Monte Carlo simulation. For the base case analysis, the expected cost of providing stress ulcer prophylaxis was $6,707 with histamine receptor-2 antagonist and $7,802 with proton pump inhibitor, resulting in a cost saving of $1,095 with histamine receptor-2 antagonist. The associated mortality probabilities were 3.819% and 3.825%, respectively, resulting in an absolute survival benefit of 0.006% with histamine receptor-2 antagonist. The primary drivers of incremental cost and survival were the assumptions surrounding ventilator-associated pneumonia and bleed. The probabilities that histamine receptor-2 antagonist was less costly and provided favorable survival were 89.4% and 55.7%, respectively. A secondary analysis assuming equal rates of C. difficile infection showed a cost saving of $908 with histamine

Past, present and future of A2A adenosine receptor antagonists in the therapy of Parkinson’s disease

PubMed Central

Armentero, Marie Therese; Pinna, Annalisa; Ferré, Sergi; Lanciego, José Luis; Müller, Christa E.; Franco, Rafael

2011-01-01

Several selective antagonists for adenosine A2A receptors (A2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson’s disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D2 and adenosine A2A receptors in the basal ganglia. At present it is believed that A2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson’s patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized anti-parkinsonian drug therapy, namely the existence of receptor (hetero)dimers/oligomers of G protein-coupled receptors, a topic currently the focus of intense debate within the scientific community. Dopamine D2 receptors (D2Rs) expressed in the striatum are known to form heteromers with A2A adenosine receptors. Thus, the development of heteromer-specific A2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs. PMID:21810444

Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans.

PubMed

Pinto, Sérgio M; Almendinger, Johann; Cabello, Juan; Hengartner, Michael O

2016-01-01

The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the "eat-me" signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.

Reinstatement of cocaine place-conditioning prevented by the peptide kappa-opioid receptor antagonist arodyn.

PubMed

Carey, A N; Borozny, K; Aldrich, J V; McLaughlin, J P

2007-08-13

Stress contributes to the reinstatement of cocaine-seeking behavior in abstinent subjects. Kappa-opioid receptor antagonists attenuate the behavioral effects of stress, potentially providing therapeutic value in treating cocaine abuse. Presently, the peptide arodyn produced long-lasting kappa-opioid receptor antagonism, suppressing kappa-opioid receptor agonist-induced antinociception at least 3 days after intracerebroventricular administration of 0.3 nmol. C57Bl/6J mice demonstrated cocaine-conditioned place preference, extinction over 3 weeks, and a subsequent reinstatement of place preference. Arodyn pretreatment suppressed stress-induced, but not cocaine-exposed, reinstatement of cocaine place preference. These results verify that arodyn and other kappa-opioid receptor antagonists may be useful therapeutics for cocaine abuse.

Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

PubMed

Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

2004-01-01

In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

Pharmacological significance of the interplay between angiotensin receptors: MAS receptors as putative final mediators of the effects elicited by angiotensin AT1 receptors antagonists.

PubMed

Pernomian, Larissa; Pernomian, Laena; Gomes, Mayara S; da Silva, Carlos H T P

2015-12-15

The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

The necessity and effectiveness of mineralocorticoid receptor antagonist in the treatment of diabetic nephropathy.

PubMed

Sato, Atsuhisa

2015-06-01

Diabetes mellitus is a major cause of chronic kidney disease (CKD), and diabetic nephropathy is the most common primary disease necessitating dialysis treatment in the world including Japan. Major guidelines for treatment of hypertension in Japan, the United States and Europe recommend the use of angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers, which suppress the renin-angiotensin system (RAS), as the antihypertensive drugs of first choice in patients with coexisting diabetes. However, even with the administration of RAS inhibitors, failure to achieve adequate anti-albuminuric, renoprotective effects and a reduction in cardiovascular events has also been reported. Inadequate blockade of aldosterone may be one of the reasons why long-term administration of RAS inhibitors may not be sufficiently effective in patients with diabetic nephropathy. This review focuses on treatment in diabetic nephropathy and discusses the significance of aldosterone blockade. In pre-nephropathy without overt nephropathy, a mineralocorticoid receptor antagonist can be used to enhance the blood pressure-lowering effects of RAS inhibitors, improve insulin resistance and prevent clinical progression of nephropathy. In CKD categories A2 and A3, the addition of a mineralocorticoid receptor antagonist to an RAS inhibitor can help to maintain 'long-term' antiproteinuric and anti-albuminuric effects. However, in category G3a and higher, sufficient attention must be paid to hyperkalemia. Mineralocorticoid receptor antagonists are not currently recommended as standard treatment in diabetic nephropathy. However, many studies have shown promise of better renoprotective effects if mineralocorticoid receptor antagonists are appropriately used.

Selective dopamine receptor 4 activation mediates the hippocampal neuronal calcium response via IP3 and ryanodine receptors.

PubMed

Wang, Ya-Li; Wang, Jian-Gang; Guo, Fang-Li; Gao, Xia-Huan; Zhao, Dan-Dan; Zhang, Lin; Wang, Jian-Zhi; Lu, Cheng-Biao

2017-09-01

Intracellular calcium is a key factor in most cellular processes, including cell growth, differentiation, proliferation and neurotransmitter release. Dopamine (DA) mediates synaptic transmission by regulating the intracellular calcium content. It is not clear, however, which specific subunit of the DA receptor contributes to DA modulation of intracellular calcium content changes. Through the traditional technique of Fura-2 calcium imaging, this study demonstrated that the DA can induce transient calcium in cultured hippocampal neurons and that this response can be mimicked by a selective dopamine receptor 4 (DR4) agonist PD168077 (PD). PD-induced calcium transience can be blocked by a calcium chelator, such as BAPTA-AM, or by pre-treatment of neurons with thapsigargin, a IP 3 receptor antagonist, or a micromolar concentration of ryanodine, a ryanodine receptor (RyR) antagonist. However PD-induced calcium transience cannot be blocked by pre-treatment of neurons with a free-calcium medium or a cocktail of NMDA receptor, L-type calcium channel and alpha7 nicotinic acetylcholine receptor blockers. These results indicate that the calcium response induced by DR4 activation is mainly through activation of IP 3 receptor in internal stores, which is likely to contribute to the DA modulation of synaptic transmission and cognitive function. Copyright © 2017. Published by Elsevier B.V.

An Autoinflammatory Disease with Deficiency of the Interleukin-1–Receptor Antagonist

PubMed Central

Aksentijevich, Ivona; Masters, Seth L.; Ferguson, Polly J.; Dancey, Paul; Frenkel, Joost; van Royen-Kerkhoff, Annet; Laxer, Ron; Tedgård, Ulf; Cowen, Edward W.; Pham, Tuyet-Hang; Booty, Matthew; Estes, Jacob D.; Sandler, Netanya G.; Plass, Nicole; Stone, Deborah L.; Turner, Maria L.; Hill, Suvimol; Butman, John A.; Schneider, Rayfel; Babyn, Paul; El-Shanti, Hatem I.; Pope, Elena; Barron, Karyl; Bing, Xinyu; Laurence, Arian; Lee, Chyi-Chia R.; Chapelle, Dawn; Clarke, Gillian I.; Ohson, Kamal; Nicholson, Marc; Gadina, Massimo; Yang, Barbara; Korman, Benjamin D.; Gregersen, Peter K.; van Hagen, P. Martin; Hak, A. Elisabeth; Huizing, Marjan; Rahman, Proton; Douek, Daniel C.; Remmers, Elaine F.; Kastner, Daniel L.; Goldbach-Mansky, Raphaela

2010-01-01

Background Autoinflammatory diseases manifest inflammation without evidence of infection, high-titer autoantibodies, or autoreactive T cells. We report a disorder caused by mutations of IL1RN, which encodes the interleukin-1–receptor antagonist, with prominent involvement of skin and bone. Methods We studied nine children from six families who had neonatal onset of sterile multifocal osteomyelitis, periostitis, and pustulosis. Response to empirical treatment with the recombinant interleukin-1–receptor antagonist anakinra in the first patient prompted us to test for the presence of mutations and changes in proteins and their function in interleukin-1–pathway genes including IL1RN. Results We identified homozygous mutations of IL1RN in nine affected children, from one family from Newfoundland, Canada, three families from the Netherlands, and one consanguineous family from Lebanon. A nonconsanguineous patient from Puerto Rico was homozygous for a genomic deletion that includes IL1RN and five other interleukin-1–family members. At least three of the mutations are founder mutations; heterozygous carriers were asymptomatic, with no cytokine abnormalities in vitro. The IL1RN mutations resulted in a truncated protein that is not secreted, thereby rendering cells hyperresponsive to interleukin-1β stimulation. Patients treated with anakinra responded rapidly. Conclusions We propose the term deficiency of the interleukin-1–receptor antagonist, or DIRA, to denote this autosomal recessive autoinflammatory disease caused by mutations affecting IL1RN. The absence of interleukin-1–receptor antagonist allows unopposed action of interleukin-1, resulting in life-threatening systemic inflammation with skin and bone involvement. (ClinicalTrials.gov number, NCT00059748.) PMID:19494218

Acetylcholine and the alpha 7 nicotinic receptor: a potential therapeutic target for the treatment of periodontal disease?

PubMed Central

2013-01-01

Objectives The aim of this review is to examine the evidence for a functional cholinergic system operating within the periodontium and determine the evidence for its role in periodontal immunity. Introduction Acetylcholine can influence the immune system via the ‘cholinergic anti-inflammatory pathway’. This pathway is mediated by the vagus nerve which releases acetylcholine to interact with the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) on proximate immuno-regulatory cells. Activation of the α7nAChR on these cells leads to down-regulated expression of pro-inflammatory mediators and thus regulates localised inflammatory responses. The role of the vagus nerve in periodontal pathophysiology is currently unknown. However, non-neuronal cells can also release acetylcholine and express the α7nAChR; these include keratinocytes, fibroblasts, T cells, B cells and macrophages. Therefore, by both autocrine and paracrine methods non-neuronal acetylcholine can also be hypothesised to modulate the localised immune response. Methods A Pubmed database search was performed for studies providing evidence for a functional cholinergic system operating in the periodontium. In addition, literature on the role of the ‘cholinergic anti-inflammatory pathway’ in modulating the immune response was extrapolated to hypothesise that similar mechanisms of immune regulation occur within the periodontium. Conclusion The evidence suggests a functional nonneuronal ‘cholinergic anti-inflammatory pathway’ may operate in the periodontium and that this may be targeted therapeutically to treat periodontal disease. PMID:22777144

Behavioral characterization of mice lacking histamine H(3) receptors.

PubMed

Toyota, Hiroshi; Dugovic, Christine; Koehl, Muriel; Laposky, Aaron D; Weber, China; Ngo, Karen; Wu, Ying; Lee, Doo Hyun; Yanai, Kazuhiko; Sakurai, Eiko; Watanabe, Takehiko; Liu, Changlu; Chen, Jingcai; Barbier, Ann J; Turek, Fred W; Fung-Leung, Wai-Ping; Lovenberg, Timothy W

2002-08-01

Brain histamine H(3) receptors are predominantly presynaptic and serve an important autoregulatory function for the release of histamine and other neurotransmitters. They have been implicated in a variety of brain functions, including arousal, locomotor activity, thermoregulation, food intake, and memory. The recent cloning of the H(3) receptor in our laboratory has made it possible to create a transgenic line of mice devoid of H(3) receptors. This paper provides the first description of the H(3) receptor-deficient mouse (H(3)(-/-)), including molecular and pharmacologic verification of the receptor deletion as well as phenotypic screens. The H(3)(-/-) mice showed a decrease in overall locomotion, wheel-running behavior, and body temperature during the dark phase but maintained normal circadian rhythmicity. H(3)(-/-) mice were insensitive to the wake-promoting effects of the H(3) receptor antagonist thioperamide. We also observed a slightly decreased stereotypic response to the dopamine releaser, methamphetamine, and an insensitivity to the amnesic effects of the cholinergic receptor antagonist, scopolamine. These data indicate that the H(3) receptor-deficient mouse represents a valuable model for studying histaminergic regulation of a variety of behaviors and neurotransmitter systems, including dopamine and acetylcholine.

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis

PubMed Central

Jenkinson, Karl M; Southwell, Bridget R; Furness, John B

1999-01-01

In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r.We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum.SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis.The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leuψ[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-βAla) were both more potent in inhibiting endocytosis (50× and 8× greater respectively) against septide than against SP.The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor. PMID:10051129

Two affinities for a single antagonist at the neuronal NK1 tachykinin receptor: evidence from quantitation of receptor endocytosis.

PubMed

Jenkinson, K M; Southwell, B R; Furness, J B

1999-01-01

1. In smooth muscle contractility assays, many NK1 receptor (NK1r) antagonists inhibit responses to the neurotransmitter, substance P (SP), and its analogue, septide, with markedly different potency, leading to the proposal that there is a septide-preferring receptor related to the NK1r. 2. We used fluorescence immunohistochemistry and confocal microscopy to visualize agonist-induced NK1r endocytosis and analyse agonist/antagonist interactions at native NK1r in neurons of the myenteric plexus of guinea-pig ileum. 3. SP and septide gave sigmoid log concentration-response curves and were equipotent in inducing NK1r endocytosis. 4. The NK1r antagonists, CP-99994 (2S,3S)-3-(2-methoxybenzyl)amino-2-phenylpiperidine dihydrochloride and MEN-10581, cyclo(Leu,[CH2NH]Lys(benzyloxycarbonyl)-Gln-Trp-Phe-betaAla) were both more potent in inhibiting endocytosis (50 x and 8 x greater respectively) against septide than against SP. 5. The results suggest that SP and septide interact differently with the NK1r, and that a single antagonist can exhibit different affinities at a single NK1r population, depending on the agonist with which it competes. Thus it may not be necessary to posit a separate septide-preferring tachykinin receptor.

Emerging growth factor receptor antagonists for the treatment of renal cell carcinoma.

PubMed

Zahoor, Haris; Rini, Brian I

2016-12-01

The landscape of systemic treatment for metastatic renal cell carcinoma (RCC) has dramatically changed with the introduction of targeted agents including vascular endothelial growth factor (VEGF) inhibitors. Recently, multiple new agents including growth factor receptor antagonists and a checkpoint inhibitor were approved for the treatment of refractory metastatic RCC based on encouraging benefit shown in clinical trials. Areas covered: The background and biological rationale of existing treatment options including a brief discussion of clinical trials which led to their approval, is presented. This is followed by reviewing the limitations of these therapeutic options, medical need to develop new treatments and major goals of ongoing research. We then discuss two recently approved growth factor receptor antagonists i.e. cabozantinib and lenvatinib, and a recently approved checkpoint inhibitor, nivolumab, and issues pertaining to drug development, and future directions in treatment of metastatic RCC. Expert opinion: Recently approved growth factor receptor antagonists have shown encouraging survival benefit but associated drug toxicity is a major issue. Nivolumab, a programmed death 1 (PD-1) checkpoint inhibitor, has similarly shown survival benefit and is well tolerated. With multiple options now available in this patient population, the right sequence of these agents remains to be determined.

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms.

PubMed

Zwart, Ruud; Reed, Hannah; Sher, Emanuele

2018-01-01

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

Loss of M5 muscarinic acetylcholine receptors leads to cerebrovascular and neuronal abnormalities and cognitive deficits in mice.

PubMed

Araya, Runa; Noguchi, Takanori; Yuhki, Munehiro; Kitamura, Naohito; Higuchi, Makoto; Saido, Takaomi C; Seki, Kenjiro; Itohara, Shigeyoshi; Kawano, Masako; Tanemura, Kentaro; Takashima, Akihiko; Yamada, Kazuyuki; Kondoh, Yasushi; Kanno, Iwao; Wess, Jürgen; Yamada, Masahisa

2006-11-01

The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R-/- mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R-/- mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R-/- mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R-/- mice. In M5R-/- mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.

Blocking α4β2 and α7 nicotinic acetylcholine receptors inhibits the reinstatement of morphine-induced CPP by drug priming in mice.

PubMed

Feng, Bin; Xing, Jiang-hao; Jia, Dong; Liu, Shui-bing; Guo, Hong-ju; Li, Xiao-qiang; He, Xiao-sheng; Zhao, Ming-gao

2011-06-20

Investigating the interaction between nicotinic and opioid receptors is of great interest for both basic mechanistic and clinical reasons. Morphine and nicotine, two common drugs of abuse, share several behavioral and rewarding properties. However, little is known about the subtypes of nicotinic acetylcholine receptors (nAChR) in the reinstatement of morphine-induced conditioned place preference (CPP). In this study, we found that a non-specific nAChR agonist, nicotine (0.5mg/kg), had no effects on the reinstatement of morphine-induced CPP. However, we found that pretreatment with specific α(4)β(2) and α(7) nAChR subtype antagonists, dihydroxy-β-erithroidine (DHβE, 5mg/kg) and methyllycaconitine (MLA, 4 mg/kg), 20 min prior to administration of morphine, inhibited the reinstatement of morphine-induced CPP by drug priming in mice. Furthermore, depression of the reinstatement of morphine-induced CPP by a single DHβE or MLA treatment lasted at least three days later when the reinstatement was induced by morphine priming. The data suggest that specific nAChR subtypes, i.e., α(4)β(2) and α(7), may contribute to the reinstatement of morphine-induced CPP by drug priming in mice. Copyright © 2011 Elsevier B.V. All rights reserved.

CCR5 receptor antagonists: discovery and SAR study of guanylhydrazone derivatives.

PubMed

Wei, Robert G; Arnaiz, Damian O; Chou, Yuo-Ling; Davey, Dave; Dunning, Laura; Lee, Wheeseong; Lu, Shou-Fu; Onuffer, James; Ye, Bin; Phillips, Gary

2007-01-01

High throughput screening (HTS) led to the identification of the guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde as a CCR5 receptor antagonist. Initial modifications of the guanylhydrazone series indicated that substitution of the benzyl group at the para-position was well tolerated. Substitution at the 5-position of the central phenyl ring was critical for potency. Replacement of the guanylhydrazone group led to the discovery of a novel series of CCR5 antagonists.

Serotonergic 5-HT6 Receptor Antagonists: Heterocyclic Chemistry and Potential Therapeutic Significance.

PubMed

Bali, Alka; Singh, Shalu

2015-01-01

The serotonin 5-HT(6) receptor (5- HT(6)R) is amongst the recently discovered serotonergic receptors with almost exclusive localization in the brain. Hence, this receptor is fast emerging as a promising target for cognition enhancement in central nervous system (CNS) diseases such as Alzheimer's disease (cognitive function), obesity, schizophrenia and anxiety. The last decade has seen a surge of literature reports on the functional role of this receptor in learning and memory processes and investigations related to the chemistry and pharmacology of 5-HT(6) receptor ligands, especially 5- HT(6) receptor antagonists. Studies show the involvement of multiple neurotransmitter systems in cognitive enhancement by 5-HT(6)R antagonists including cholinergic, glutamatergic, and GABAergic systems. Several of the 5-HT(6)R ligands are indole based agents bearing structural similarity to the endogenous neurotransmitter serotonin. Based on the pharmacophoric models proposed for these agents, drug designing has been carried out incorporating various heterocyclic replacements for the indole nucleus. In this review, we have broadly summarized the medicinal chemistry and current status of this fairly recent class of drugs along with their potential therapeutic applications.

Classification and virtual screening of androgen receptor antagonists.

PubMed

Li, Jiazhong; Gramatica, Paola

2010-05-24

Computational tools, such as quantitative structure-activity relationship (QSAR), are highly useful as screening support for prioritization of substances of very high concern (SVHC). From the practical point of view, QSAR models should be effective to pick out more active rather than inactive compounds, expressed as sensitivity in classification works. This research investigates the classification of a big data set of endocrine-disrupting chemicals (EDCs)-androgen receptor (AR) antagonists, mainly aiming to improve the external sensitivity and to screen for potential AR binders. The kNN, lazy IB1, and ADTree methods and the consensus approach were used to build different models, which improve the sensitivity on external chemicals from 57.1% (literature) to 76.4%. Additionally, the models' predictive abilities were further validated on a blind collected data set (sensitivity: 85.7%). Then the proposed classifiers were used: (i) to distinguish a set of AR binders into antagonists and agonists; (ii) to screen a combined estrogen receptor binder database to find out possible chemicals that can bind to both AR and ER; and (iii) to virtually screen our in-house environmental chemical database. The in silico screening results suggest: (i) that some compounds can affect the normal endocrine system through a complex mechanism binding both to ER and AR; (ii) new EDCs, which are nonER binders, but can in silico bind to AR, are recognized; and (iii) about 20% of compounds in a big data set of environmental chemicals are predicted as new AR antagonists. The priority should be given to them to experimentally test the binding activities with AR.

Crosstalk between beta-2-adrenoceptor and muscarinic acetylcholine receptors in the airway.

PubMed

Pera, Tonio; Penn, Raymond B

2014-06-01

The M3 and M2 muscarinic acetylcholine receptors (mAChRs) and beta-2-adrenoceptors (β2ARs) are important regulators of airway cell function, and drugs targeting these receptors are among the first line drugs in the treatment of the obstructive lung diseases asthma and chronic obstructive lung disease (COPD). Cross-regulation or crosstalk between mAChRs and β2ARs in airway smooth muscle (ASM) helps determine the contractile state of the muscle, thus airway diameter and resistance to airflow. In this review we will detail mAChR and β2AR-signaling and crosstalk, focusing on events in the ASM cell but also addressing the function of these receptors in other cell types that impact airway physiology. We conclude by discussing how recent advances in GPCR pharmacology offer a unique opportunity to fine tune mAChR and β2AR signaling and their crosstalk, and thereby produce superior therapeutics for obstructive lung and other diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Activation of muscle nicotinic acetylcholine receptor channels by nicotinic and muscarinic agonists

PubMed Central

Akk, Gustav; Auerbach, Anthony

1999-01-01

The dose-response parameters of recombinant mouse adult neuromuscular acetylcholine receptor channels (nAChR) activated by carbamylcholine, nicotine, muscarine and oxotremorine were measured. Rate constants for agonist association and dissociation, and channel opening and closing, were estimated from single-channel kinetic analysis.The dissociation equilibrium constants were (mM): ACh (0.16)carbamylcholine (5.1)>oxotremorine M (0.6)>nicotine (0.5)>muscarine (0.15).Rat neuronal α4β2 nAChR can be activated by all of the agonists. However, detailed kinetic analysis was impossible because the recordings lacked clusters representing the activity of a single receptor complex. Thus, the number of channels in the patch was unknown and the activation rate constants could not be determined.Considering both receptor affinity and agonist efficacy, muscarine and oxotremorine are significant agonists of muscle-type nAChR. The results are discussed in terms of structure-function relationships at the nAChR transmitter binding site. PMID:10602325

A Quorum-Sensing Antagonist Targets Both Membrane-Bound and Cytoplasmic Receptors And Controls Bacterial Pathogenicity

PubMed Central

Swem, Lee R.; Swem, Danielle L.; O’Loughlin, Colleen T.; Gatmaitan, Raleene; Zhao, Bixiao; Ulrich, Scott M.; Bassler, Bonnie L.

2009-01-01

Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development. PMID:19647512

Computational determination of the binding mode of α-conotoxin to nicotinic acetylcholine receptor

NASA Astrophysics Data System (ADS)

Tabassum, Nargis; Yu, Rilei; Jiang, Tao

2016-12-01

Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The α-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, α-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of α-conotoxins in complex with acetylcholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the α1 and α9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of α-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of α-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between α-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of α-conotoxins on AChRs allows rational design of α-conotoxin analogues with improved potency or selectivity to nAChRs.

An assessment of the effects of serotonin 6 (5-HT6) receptor antagonists in rodent models of learning.

PubMed

Lindner, Mark D; Hodges, Donald B; Hogan, John B; Orie, Anitra F; Corsa, Jason A; Barten, Donna M; Polson, Craig; Robertson, Barbara J; Guss, Valerie L; Gillman, Kevin W; Starrett, John E; Gribkoff, Valentin K

2003-11-01

Antagonists of serotonin 6 (5-HT6) receptors have been reported to enhance cognition in animal models of learning, although this finding has not been universal. We have assessed the therapeutic potential of the specific 5-HT6 receptor antagonists 4-amino-N-(2,6-bis-methylamino-pyrimidin-4-yl)-benzenesulfonamide (Ro 04-6790) and 5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide (SB-271046) in rodent models of cognitive function. Although mice express the 5-HT6 receptor and the function of this receptor has been investigated in mice, all reports of activity with 5-HT6 receptor antagonists have used rat models. In the present study, receptor binding revealed that the pharmacological properties of the mouse receptor are different from the rat and human receptor: Ro 04-6790 does not bind to the mouse 5-HT6 receptor, so all in vivo testing included in the present report was conducted in rats. We replicated previous reports that 5-HT6 receptor antagonists produce a stretching syndrome previously shown to be mediated through cholinergic mechanisms, but Ro 04-6790 and SB-271046 failed to attenuate scopolamine-induced deficits in a test of contextual fear conditioning. We also failed to replicate the significant effects reported previously in both an autoshaping task and in a version of the Morris water maze. The results of our experiments are not consistent with previous reports that suggested that 5-HT6 antagonists might have therapeutic potential for cognitive disorders.

Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

PubMed

Lundstrom, K; Hawcock, A B; Vargas, A; Ward, P; Thomas, P; Naylor, A

1997-10-15

Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

The cardiac sympathetic co-transmitter galanin reduces acetylcholine release and vagal bradycardia: Implications for neural control of cardiac excitability

PubMed Central

Herring, Neil; Cranley, James; Lokale, Michael N.; Li, Dan; Shanks, Julia; Alston, Eric N.; Girard, Beatrice M.; Carter, Emma; Parsons, Rodney L.; Habecker, Beth A.; Paterson, David J.

2012-01-01

The autonomic phenotype of congestive cardiac failure is characterised by high sympathetic drive and impaired vagal tone, which are independent predictors of mortality. We hypothesize that impaired bradycardia to peripheral vagal stimulation following high-level sympathetic drive is due to sympatho-vagal crosstalk by the adrenergic co-transmitters galanin and neuropeptide-Y (NPY). Moreover we hypothesize that galanin acts similarly to NPY by reducing vagal acetylcholine release via a receptor mediated, protein kinase-dependent pathway. Prolonged right stellate ganglion stimulation (10 Hz, 2 min, in the presence of 10 μM metoprolol) in an isolated guinea pig atrial preparation with dual autonomic innervation leads to a significant (p < 0.05) reduction in the magnitude of vagal bradycardia (5 Hz) maintained over the subsequent 20 min (n = 6). Immunohistochemistry demonstrated the presence of galanin in a small number of tyrosine hydroxylase positive neurons from freshly dissected stellate ganglion tissue sections. Following 3 days of tissue culture however, most stellate neurons expressed galanin. Stellate stimulation caused the release of low levels of galanin and significantly higher levels of NPY into the surrounding perfusate (n = 6, using ELISA). The reduction in vagal bradycardia post sympathetic stimulation was partially reversed by the galanin receptor antagonist M40 after 10 min (1 μM, n = 5), and completely reversed with the NPY Y2 receptor antagonist BIIE 0246 at all time points (1 μM, n = 6). Exogenous galanin (n = 6, 50–500 nM) also reduced the heart rate response to vagal stimulation but had no effect on the response to carbamylcholine that produced similar degrees of bradycardia (n = 6). Galanin (500 nM) also significantly attenuated the release of 3H-acetylcholine from isolated atria during field stimulation (5 Hz, n = 5). The effect of galanin on vagal bradycardia could be abolished by the galanin receptor antagonist

D2 dopamine receptor activation inhibits basal and forskolin-evoked acetylcholine release from dissociated striatal cholinergic interneurons.

PubMed

Login, I S

1997-02-21

We tested whether D2 ligands inhibit basal and forskolin-stimulated [3H]ACh release from dissociated striata, as opposed to striatal slices. Quinpirole inhibited both basal (40% maximal inhibition; IC50 approximately 50 nM) and 10 microM forskolin-stimulated release (80% inhibition; IC50 approximately 25 nM quinpirole) and both actions were blocked by a D2 antagonist. Vesamicol prevented the quinpirole and forskolin actions. The ability of D2 agonists to inhibit basal and cyclase-stimulated acetylcholine release emanating from vesamicol-sensitive vesicles appears to be tonically suppressed by inhibitory elements within striatal circuitry.

NMDA receptor antagonists extend the sensitive period for imprinting.

PubMed

Parsons, C H; Rogers, L J

2000-03-01

Filial imprinting in the domestic chick occurs during a sensitive period of development. The exact timing of this period can vary according to the methods used to measure imprinting. Using our imprinting paradigm, we have shown that normal, dark-reared chicks lose the ability to imprint after the second day post-hatching. Further, we reported that chicks treated 10 h after hatching with a mixture of the noncompetitive NMDA receptor antagonist ketamine (55 mg/kg) and the alpha(2)-adrenergic receptor agonist xylazine (6 mg/kg) were able to imprint on day 8 after hatching, whereas controls treated with saline did not imprint. We now show that the effect of the ketamine-xylazine mixture can be mimicked by treating chicks with ketamine alone or with another noncompetitive NMDA receptor antagonist, MK-801 (5 mg/kg). Treating chicks with a single dose of ketamine (55 mg/kg) or with a single dose of xylazine (6 mg/kg) failed to produce the effect on the sensitive period. However, prolonging the action of ketamine by treating chicks with two doses of ketamine (at 10 and 12 h after hatching) did allow imprinting on day 8. In contrast, prolonging the action of xylazine had no effect on the sensitive period for imprinting. Chicks treated with MK-801 were also able to imprint on day 8. Thus, we have evidence that the NMDA receptor system is involved in the mechanisms that control the sensitive period for imprinting.

Cholecystokinin receptor antagonist halts progression of pancreatic cancer precursor lesions and fibrosis in mice.

PubMed

Smith, Jill P; Cooper, Timothy K; McGovern, Christopher O; Gilius, Evan L; Zhong, Qing; Liao, Jiangang; Molinolo, Alfredo A; Gutkind, J Silvio; Matters, Gail L

2014-10-01

Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy. Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001). These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

CHOLECYSTOKININ RECEPTOR ANTAGONIST HALTS PROGRESSION OF PANCREATIC CANCER PRECURSOR LESIONS AND FIBROSIS IN MICE

PubMed Central

Smith, Jill P.; Cooper, Timothy K.; McGovern, Christopher O.; Gilius, Evan L.; Zhong, Qing; Liao, Jiangang; Molinolo, Alfredo A.; Gutkind, J. Silvio; Matters, Gail L.

2014-01-01

Objectives Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved with the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer. Methods The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-KrasG12D transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK-receptor antagonist (proglumide, 0.1mg/ml). Pancreas from mice were removed and examined histologically for number and grade of PanINs after 1, 2 or 4 months of antagonist therapy. Results Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed and progression to advanced lesions arrested in mice treated with proglumide compared to controls (p=0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared to vehicle (pitalic>0.001). Conclusions These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. Use of CCK-receptor antagonists may have a role in cancer prophylaxis in high risk subjects, and may reduce fibrosis in the microenvironment. PMID:25058882

Nonconventional three-finger toxin BMLCL from krait Bungarus multicinctus venom with high affinity interacts with nicotinic acetylcholine receptors.

PubMed

Utkin, Yu N; Kasheverov, I E; Kudryavtsev, D S; Andreeva, T V; Starkov, V G; Ziganshin, R H; Kuznetsov, D V; Anh, Hoang Ngoc; Thao, Nguyen Thi Thanh; Khoa, Nguyen Cuu; Tsetlin, V I

2015-01-01

Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

PubMed Central

Dennis, Siobhan H.; Pasqui, Francesca; Colvin, Ellen M.; Sanger, Helen; Mogg, Adrian J.; Felder, Christian C.; Broad, Lisa M.; Fitzjohn, Steve M.; Isaac, John T.R.; Mellor, Jack R.

2016-01-01

Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks

PubMed Central

Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.

2003-01-01

Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539

Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaplan, I.; Blakely, B.T.; Pavlath, G.K.

1990-10-01

Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immunemore » system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.« less

Tachykinin-mediated respiratory effects in conscious guinea pigs: modulation by NK1 and NK2 receptor antagonists.

PubMed

Kudlacz, E M; Logan, D E; Shatzer, S A; Farrell, A M; Baugh, L E

1993-09-07

Tachykinins, in particular neurokinin A and substance P, produce a number of airway effects which may contribute to respiratory diseases such as asthma. We examined the ability of aerosolized substance P, neurokinin A or capsaicin to produce respiratory alterations in conscious guinea pigs using modified whole body plethysmography. Substance P-mediated dyspnea and significant respiratory events were inhibited by the NK1 receptor antagonist, CP-96,345. Neurokinin A-mediated respiratory effects were ablated by the NK2 receptor antagonists: MEN 10207, MDL 29,913 and SR 48,968, the latter being the most potent. The peptide-based antagonist, MEN 10207, produced respiratory effects itself suggesting partial agonist activity. The cyclic hexapeptide, MDL 29,913, relaxed airway smooth muscle via mechanisms other than tachykinin antagonism. NK2 but not NK1 receptor antagonists were able to delay the onset of capsaicin-induced dyspnea, although alone they did not usually (in approximately 10% of the animals) eliminate the response. However, when NK2 receptor antagonists were combined with CP-96,345, the incidence of dyspnea induced by capsaicin decreased significantly (40%) suggesting that both tachykinins contribute to dyspnea in this system.

Test-retest paradigm of the forced swimming test in female mice is not valid for predicting antidepressant-like activity: participation of acetylcholine and sigma-1 receptors.

PubMed

Su, Jing; Hato-Yamada, Noriko; Araki, Hiroaki; Yoshimura, Hiroyuki

2013-01-01

The forced swimming test (FST) in mice is widely used to predict the antidepressant activity of a drug, but information describing the immobility of female mice is limited. We investigated whether a prior swimming experience affects the immobility duration in a second FST in female mice and whether the test-retest paradigm is a valid screening tool for antidepressants. Female ICR mice were exposed to the FST using two experimental paradigms: a single FST and a double FST in which mice had experienced FST once 24 h prior to the second trail. The initial FST experience reliably prolonged immobility duration in the second FST. The antidepressants imipramine and paroxetine significantly reduced immobility duration in the single FST, but not in the double FST. Scopolamine and the sigma-1 (σ1) antagonist NE-100 administered before the second trial significantly prevented the prolongation of immobility. Neither a 5-HT1A nor a 5-HT2A receptor agonist affected immobility duration. We suggest that the test-retest paradigm in female mice is not adequate for predicting antidepressant-like activity of a drug; the prolongation of immobility in the double FST is modulated through acetylcholine and σ1 receptors.

Preclinical and clinical characterization of the selective 5-HT(1A) receptor antagonist DU-125530 for antidepressant treatment.

PubMed

Scorza, M C; Lladó-Pelfort, L; Oller, S; Cortés, R; Puigdemont, D; Portella, M J; Pérez-Egea, R; Alvarez, E; Celada, P; Pérez, V; Artigas, F

2012-11-01

The antidepressant efficacy of selective 5-HT reuptake inhibitors (SSRI) and other 5-HT-enhancing drugs is compromised by a negative feedback mechanism involving 5-HT(1A) autoreceptor activation by the excess 5-HT produced by these drugs in the somatodendritic region of 5-HT neurones. 5-HT(1A) receptor antagonists augment antidepressant-like effects in rodents by preventing this negative feedback, and the mixed β-adrenoceptor/5-HT(1A) receptor antagonist pindolol improves clinical antidepressant effects by preferentially interacting with 5-HT(1A) autoreceptors. However, it is unclear whether 5-HT(1A) receptor antagonists not discriminating between pre- and post-synaptic 5-HT(1A) receptors would be clinically effective. We characterized the pharmacological properties of the 5-HT(1A) receptor antagonist DU-125530 using receptor autoradiography, intracerebral microdialysis and electrophysiological recordings. Its capacity to accelerate/enhance the clinical effects of fluoxetine was assessed in a double-blind, randomized, 6 week placebo-controlled trial in 50 patients with major depression (clinicaltrials.gov identifier NCT01119430). DU-125530 showed equal (low nM) potency to displace agonist and antagonist binding to pre- and post-synaptic 5-HT(1A) receptors in rat and human brain. It antagonized suppression of 5-hydroxytryptaminergic activity evoked by 8-OH-DPAT and SSRIs in vivo. DU-125530 augmented SSRI-induced increases in extracellular 5-HT as effectively as in mice lacking 5-HT(1A) receptors, indicating a silent, maximal occupancy of pre-synaptic 5-HT(1A) receptors at the dose used. However, DU-125530 addition to fluoxetine did not accelerate nor augment its antidepressant effects. DU-125530 is an excellent pre- and post-synaptic 5-HT(1A) receptor antagonist. However, blockade of post-synaptic 5- HT(1A) receptors by DU-125530 cancels benefits obtained by enhancing pre-synaptic 5-hydroxytryptaminergic function. © 2011 The Authors. British Journal of

Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR*

PubMed Central

Li, Hao; Redinbo, Matthew R.; Venkatesh, Madhukumar; Ekins, Sean; Chaudhry, Anik; Bloch, Nicolin; Negassa, Abdissa; Mukherjee, Paromita; Kalpana, Ganjam; Mani, Sridhar

2013-01-01

The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple “gain-of-function” mutants that were “resistant” to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications. PMID:23525103

Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bray, J.J.; Drachman, D.B.

1982-01-01

Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the rangemore » of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.« less

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes.

PubMed Central

Maggi, C. A.; Patacchini, R.; Giuliani, S.; Rovero, P.; Dion, S.; Regoli, D.; Giachetti, A.; Meli, A.

1990-01-01

1. We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium-denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2. In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 3. The peptide antagonists tested were: Peptide I = [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10); Peptide II = [Tyr5, D-Trp6,8,9, Arg10]-NKA(3-10); Peptide III = Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2. The three peptides produced a concentration-dependent rightward shift of the concentration-response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4. The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [beta Ala8]-NKA(4-10) was used instead of NKA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2167737

Distribution of efferent cholinergic terminals and alpha-bungarotoxin binding to putative nicotinic acetylcholine receptors in the human vestibular end-organs.

PubMed

Ishiyama, A; Lopez, I; Wackym, P A

1995-11-01

Although acetylcholine (ACh) has been identified as the primary neurotransmitter of the efferent vestibular system in most animals studied, no direct evidence exists that ACh is the efferent neurotransmitter of the human vestibular system. Choline acetyltransferase immunohistochemistry (ChATi), acetylcholinesterase (AChE) histochemistry, and alpha-bungarotoxin binding were used in human vestibular end-organs to address this question. ChATi and AChE activity was found in numerous bouton-type terminals contacting the basal area of type II vestibular hair cells and the afferent chalices surrounding type I hair cells; alpha-bungarotoxin binding suggested the presence of nicotinic acetylcholine receptors on type II vestibular hair cells and on the afferent chalices surrounding type I hair cells. This study provides evidence that the human efferent vestibular axons and terminals are cholinergic and that the receptors receiving this innervation may be nicotinic.

Decrement in operant performance produced by NMDA receptor antagonists in the rat: tolerance and cross-tolerance.

PubMed

Dravolina, O A; Zvartau, E E; Bespalov, A Y

2000-04-01

Current perspectives on the clinical use of NMDA receptor antagonists infer repeated administration schedules for the management of different pathological states. The development of tolerance and cross-tolerance between different NMDA receptor antagonists may be an important factor contributing to the clinical efficacy of these drugs. The present study aimed to characterize the development of tolerance and cross-tolerance to the ability of various site-selective NMDA receptor antagonists to produce a decrement of operant responding (multiple extinction 9 s fixed-interval 1-s schedule of water reinforcement). Acute administration of D-CPPen (SDZ EAA 494; 1-5.6 mg/kg), dizocilpine (MK-801; 0.03-0.3 mg/kg), memantine (0.3-17 mg/kg), ACEA-1021 (10-56 mg/kg), and eliprodil (1-30 mg/kg) differentially affected operant responding. Both increases and decreases in response rates and accuracy of responding were observed. Repeated preexposure to D-CPPen (5.6 mg/kg, once a day for 7 days) attenuated a behavioral disruption produced by an acute challenge with D-CPPen or ACEA-1021, but potentiated the effects of dizocilpine, memantine, and eliprodil. Based on the present results, one can suggest that the repeated administration of a competitive NMDA receptor antagonist differentially affects the functional activity of various sites on NMDA receptor complex.